.0^ 

ISF 263 

R713 
iCopy 1 



Issued April 8, 1913. 



U. S. DEPARTMENT OF AGRICULTURE, 

BUREAU OF ANIMAL INDUSTRY.— Bulletin 162. 

A. D. MELVIN, Chief of Bureau. 



FACTORS INFLUENCING THE CHANGE IN 
FLAVOR IN STORAGE BUTTER. 



BY 



L. A. ROGERS, Bacteriologist; W. N. BERG, Chemist; 
C. R. POTTEIGER, Assistant Chemist, 

AND 
B. J. DAVIS, Assistant. 




WASHINGTON: 

GOVERNMENT PRINTING OFFICE. 

1913. 



THE BUREAU OF ANIMAL INDUSTRY. 



Chief: A. D. Melvin. 

Assistant Chief: A. M. Farrington. 

Chief Clerk: Charles C. Carroll. 

Animal Husbandry Division: George M. Rommel, chief. 

Biochemic Division: M. Dorset, chief. 

Dairy Division: B. H. Rawl, chief. 

Field Inspection Division: R. A. Ramsay, chief. 

Meat Inspection Division: R. P. Steddom, chief. 

Pathological Division: John R. Mohler, chief. 

Quarantine Division: Richard W. Hickman, chief. 

Zoological Division: B. H. Ransom, chief. 

Experiment Station: E. C. Schroeder, superintendent. 

Editor: James M. Pickens. 

DAIRY DIVISION. v 

B. H. Rawl, chief. 

Helmer Rabild, in charge of Dairy Farming Investigations. 

S. C. Thompson, in charge of Dairy Manufacturing Investigations. 

L. A. Rogers, in charge of Research Laboratories. 

Ernest Kelly, in charge of Marl-et Milk Investigations. 

Robert McAdam, in charge of Renovated Butter Inspection. 



Issued April 8, 1913. 

U. S. DEPARTMENT OF AGRICULTURE, 

BUREAU OF ANIMAL INDUSTRY.— Bulletin 162. 

A. D. MF.LVIN, Chief of Bureau. 



FACTORS INFLUENCING THE CHANGE IN 
FLAVOR IN STORAGE BUTTER. 



BY 



L. A. ROGERS, Bacteriologist; W. N. BERG, Chemist; 
C. R. POTTEIGER, Assistant Chemist, 

AND 
B. J. DAVIS, Assisia?it. 




WASHINGTON: 

GOVERNMENT PRINTING OFFICE. 
1913. 






v< 



oc 



^0 



v"" 






.ETTER OF TRANSMITTAL. 



U. S. Department of Agriculture, 

Bureau of Animal Industry, 
Washington, D. C, October 8, 1912. 
Sir: I have the honor to transmit herewith for pubhcation in the 
bulletin series of this bureau a manuscript entitled "Factors Influ- 
encing the Change in Flavor in Storage Butter," b}^ Messrs. L. A. 
Rogers, W. N. Berg, C. R. Potteiger, and B. J. Davis, of the Dairy 
Division. 

Respectfully, 

A. D. Melvin, 

Chief of Bureau. 
Hon. James Wilson, 

Secretary of Agriculture. 



D. OF D. 
APB 1? 1913 



CONTENTS 



Page. 

Introduction 5 

Possible causes of change 6 

Proteolysis in butter 9 

Previous work 9 

Analytical difficulties 9 

Results by Gray and others 11 

Objections to ferric chlorid and tannic acid as protein precipitants 14 

New method for detecting proteolysis in butter 16 

The influence of sodium chlorid on the precipitability of casein by 

acetic acid , 1(5 

Method for the estimation of water-soluble nitrogen in butter 18 

Description of samples 23 

Discussion of results, Table 2 25 

Conclusions 25 

Proteolysis in milk 26 

Possible objection to the new method for detecting proteolysis in butter. . . 26 
The inhibiting effect of sodium chlorid and cold storage upon the acti\dty 

of galactase in buttermilk 27 

Method of measuring the activity of galactase in buttermilk 28 

Results 28 

The inhibiting effect of sodium chlorid and cold storage upon the activities 

of proteolytic enzyms in sterilized skim milk 30 

Description of samples 30 

Results 31 

Conclusions 31 

The indirect action of bacteria 32 

Reinoculation of cream 33 

The possible oxidation of butter by inc^losed air 34 

Method of gas analysis 35 

Results 36 

The effect of metals on butter 38 

Earlier investigations 38 

Method of analysis 42 

Relation of iron in butter to iron in the cream 43 

Distribution of iron between fat and curd solution 44 

The influence of iron on flavor 45 

The influence of copper on flavor 48 

Contamination of cream with iron from containers 50 

Theoretical considerations 55 

The oxidation of lactose in butter 57 

Description of samples ^ 58 

Methods and experimental procedure 58 

The possible oxidation of lactose in storage butter by a peroxid 61 

Odors produced in milk by the addition of iron salts 64 

The production of iodoform-reacting substances in milk by ferrous iron 66 

Summary 68 

3 



ILLUSTRATION. 



Page. 
Fig. 1. — Apparatus used for obtaining the gas from a can of butter 35 

4 



FACTORS INFLUENCING THE CHANGE IN FLAVOR IN 
STORAGE BUTTER. 



INTRODUCTION. 

The economic conditions in this country which have made it neces- 
sary to hold butter in storage for long periods have increased the 
importance of the changes that take place in butter on standmg. 
A change that passes unnoticed in butter that is used when a week or 
two old may become a serious defect after three or four months in 
storage. The great variation and complexity of the changes in flavor 
indicate a corresponding complexity in the chemical alteration in the 
butter, and while it is true that some of the modifications are well 
known it is becoming evident that the various flavors are produced 
by changes too small to be measured by the ordinary methods of the 
laboratory. Under certain circumstances free fatty acids may be 
formed, a condition usually associated with a rancid flavor. How- 
ever, it is evident that the fatty acids alone are not the cause of the 
rancid flavor, since, in the process of renovating, the rancid flavor 
is removed while a large part of the acid remains. 

It is possible that the flavor-giving substances are produced in very 
small quantities and that their formation is not necessarily comiected 
with or in proportion to the grosser changes measurable by the ordi- 
nary analytical methods. There are several substances in butter 
that are more or less unstable under ordinary circumstances, i. e., the 
proteins of milk in their hydrated condition, lecithin, citric acid, lac- 
tic acid, and other products of bacterial action. But little work has 
been done in which the storage flavor was shown to be related to 
chemical changes involving any of these substances. This is proba- 
bly due to the fact that while butter fat is easily handled for analyti- 
cal purposes, it is difficult to separate from the butter fat the other 
fatlike substances, such as lecithin. The remaining part of the 
butter, which will be called the butter curd solution, is of such a physi- 
cal consistency that it can not very well be used directly for quanti- 
tative analytical work. 

In considering the problem of storage flavor, its causes, and the 
methods of studying the problem, it is well to bear in mind one or 
two of the facts involved in the physiology of the senses of taste and 
smell. It is well known that several different substances may taste 
alike: Thus sugar, saccharin, lead acetate, glycerin, and perhaps 
still other substances, all taste sweet. Chemically they are not at 

5 



6 CHANGE IN FLAVOE OF STORAGE BUTTER. 

all similar. While trimethylamin may be the specific cause of fishy 
flavor in herring brine, it is not necessarily the cause of fishy flavor 
in butter. It is possible, reasoning by analogy, that many different 
substances may cause "fishy" flavor. 

The sense of smell is very delicate and can detect astonishingly 
small amounts of material, so small that the most delicate balances 
could not weigh them. A flavor is a mixed sensation in which the 
sense of taste and smell take a leading part. Howell * states that 
0.00005 grams of quinine in 100 cubic centimeters of water is detecti- 
ble on the root of the tongue. "It is recognized in chemical work, 
for instance, that traces of known substances too small to give the 
ordinary chemical reactions may be detected easily by the sense of 
smell. According to the experiments of Fischer and Penzoldt, mer- 
captan may be detected in a dilution of 460,000.600 of ^ milligram 
in 50 cubic centimeters of air." ^ 

While the off flavors of butter may not be caused by the formation 
of such inconceivably small amounts of odoriferous substances, yet 
such data are of practical significance in so far as they indicate that 
the analytical method of studying storage flavors may be wholly 
inadequate. There may be many substances ^ whose isolation or 
detection in butter might be very difficult if not impossible by present 
methods, and which would still impart to the butter sufficient odor 
and taste to be distinctly perceptible. 

POSSIBLE CAUSES OF CHANGE. 

The marked influence of bacteria on the flavor of milk, cheese, and 
other dairy products naturally leads to the conclusion that the same 
organisms would be an important if not the only factor concerned in 
the changes in butter. It has been demonstrated, particularly by the 
work of Jensen,^ that under certain conditions bacteria multiply in 
butter and have a direct influence on the flavor of the product. 

It should be remembered, however, that the butter on which 
Jensen and other European investigators worked differs in one very 
essential particular from the ordinary American butter. While the 
salt content of most European butter is low enough to permit the 
growth of bacteria, American butter contains suflficient salt to bring 
its concentration in the water of the butter to 18 per cent or more. 
It is to be expected that bacteria would not grow — or at least would 

1 Howell, William H. Textbook of Physiology. Philadelphia, 1906. See pp. 275-280. 

2 Zwaardemaker, H. Geruch. Ergebnisse der Physiologie. Abteilung 2, vol. 1, pp. 89&-909. Wies- 
baden, 1902. 

3 Jensen, Orla. Bakteriologische Studien Uber danische Butter. Centralblatt fiir Bakteriologie, Para- 
siteiikunde und Infektionskrankheiten. Abteilung 2, vol. 29, no. 23/25, pp. 610-61G. Jena, Apr. 8, 1911. 

Jensen, Orla. Studien iiber das Ranzigwerden der Butter. Centralblatt fiir Bakteriologie. Parasi- 
tenkunde und Infektionskrankheiten. Abteilung 2, vol. 8, no. 1, pp. 11-16, Jan. 4; no. 5, pp. 140-144, Feb. 
5; no. 6, pp. 171-174, Feb. 10; no. 7, pp. 211-216, Feb. 17; no. 8, pp. 248-252, Feb. 24; no. 9, pp. 278-281, 
Mar. 4; no. 10, pp. 309-312, Mar. 8; no. 11, pp. 342-346, Mar. 13; no. 12, pp. 367-369, Mar. 15; no. 13, pp. 
400-409, Mar. 24. Jena, 1902. 



POSSIBLE CAUSES OF CHANGE, 7 

grow only very sparsely — under these conditions, and the investiga- 
tions in this country confirm this supposition. Rahn, Brown, and 
Smith^ found in some samples of butter a torula able to grow very slowly 
in salt solutions at low temperatures, but this occurred in such small 
numbers that it could not account for much of the deterioration of 
the butter. In our own work we have found no evidence of bacterial 
growth under normal conditions, with the exception of a small multi- 
plication of torula at high storage temperatures. In these cases 
there was no apparent relation between the growth of torula and 
change in flavor. Moreover, the same changes took place in dupli- 
cate lots of butter held at temperatures so far below the freezing 
point that there could be no possibility of growth. Any flavors that 
appear in butter may be found in butter held at the commercial 
storage temperature of zero or below (Fahrenheit), and any explana- 
tion of the cause of these changes which does not take this fact into 
consideration is obviously fallacious, or at best vahd for certain con- 
ditions only. 

In some of our earlier work^ the possible influence of lipolytic 
enzyms was suggested, but it was soon found that in many cases 
butter showed a marked change in flavor without any appreciable 
hydrolysis of the fat. This observation is confirmod by the work of 
Rahn, Brown, and Smith cited above. The action of other enzyms, 
as, for mstance, the proteolytic enzym of the milk or those secreted 
by bacteria, is not necessarily excluded. 

The mfluence of the acidity of the cream on the flavor of butter has 
already been pointed out.^ It has also been suggested * that a slow 
oxidation may take place in the ulterior of a package of butter, due 
to the numerous small bubbles of air inclosed in the butter. Even a 
superficial examination of the work already done shows that the ques- 
tion is a very complicated one and that the difficulties in the way of a 
solution are many. In studymg the ripening of cheese pronounced 
chemical changes are available for measuring the progress of the ripen- 
ing. In butter the changes are scarcely appreciable. The investi- 
gator is thus forced to rely on the sense of taste and smell for a 
measure of the change. Some butter judges have developed marked 
ability m detecting and estmiatin^ the intensity of various flavors, 
but at best the sense of taste is uncertain, and any numerical scale 
based on this faculty is necessarily an arbitrary one and subject to 
fluctuation in its value. Two butter judges can not be expected 

1 Rahn, Otto, Brown, C. W., and Smith, L. M. Keeping qualities of butter. Michigan Agricultural 
College Experiment Station, Technical Bulletin 2. East Lansing, September, 1909. 

2 Rogers, Lore A. Studies upon the keeping quality of butter. United States Department of Agricul- 
ture, Bureau of Animal Industry, Bulletin 57, "Washington, 1904. 

3 Rogers, L. A., and Gray, C.E. The influence ofacidityofcream on the flavor of butter. United States 
Department of Agriculture, Bureau of Animal Industry, Bulletin 114, Washington, 1909. 

* Rogers, L. A. Fishy flavor in butter. United States Department of Agriculture, Bureau of Animal 
Industry, Circular 146, Washington, 1909. 



8 CHANGE IN FLAVOR OF STORAGE BUTT-ER. 

always to agree, because the definitions of flavors can not be reduced 
to exact terms and the amount of deduction on the numerical scale 
for various flavors can not be fixed. 

The most serious difficulty in experimental work on butter is in 
controlling the conditions under which the butter is made. So many 
apparently unimportant factors have an influence on the flavor that 
it is nearly impossible to make butter with a normal flavor and have 
only one varying factor. The work is further complicated by the 
sequence of flavors that frequently occurs in butter held in storage. 
It is evident that the usual off flavors are in many cases a combination 
of flavors and that the flavors themselves are caused by a combination 
of circumstances and not by a single cause. It is probable also that 
identical flavors may be caused by different factors. 

In the work reported in this paper we have attempted to determine 
the part plaj^ed by certain factors in the general change in flavor in 
storage butter -without directing the investigation toward any par- 
ticular flavor or attempting to cover all of the causes of deterioration. 

In this we have been guided by the previous work, which has indi- 
cated certain points at which the problem could be attacked with 
some promise of positive results. It has been observed, for instance, 
that when a lot of sweet cream is divided, one half churned at once 
and the other half pasteurized and churned, the butter from the 
unpasteurized half deteriorates very quickly, while the pasteurized- 
cream butter has exceptionally good keeping qualities. What has 
been removed by the pasteurization that has such a marked influence 
on the butter? The enzyms of the milk are partly or entirely 
destroyed and a large proportion of the bacteria are killed. Are the 
proteolytic enzyms of the milk able to work under the conditions 
existing in butter and have they any influence on the flavor of the 
butter? Is there any appreciable proteolysis in butter even under 
favorable conditions ? If the two lots of cream are ripened, the keep- 
ing quality of the butter from the unpasteurized cream is increased, 
while that from the pasteurized cream is decreased. In the process 
of ripening the bacterial growth is confined almost entirely to one 
variety, T3ut it does not necessarily follow that these bacteria have 
any direct deleterious action. The growth of the bacteria produces a 
considerable quantity of acid, and the chemical instabihty of the 
product is increased accordingly. 

Does the air which, as has been shown, is inclosed in the butter 
effect an appreciable oxidation ? Milk and cream is handled in con- 
tainers in which it may be exposed to tin, iron, or copper. Under 
these conditions it is reasonable to suppose that small amounts of the 
metals, especially the iron and copper, will be dissolved and carried 
into the butter. Do the salts formed by the metals with organic acids 
of the cream affect the flavor of the butter ? 



PROTEOLYSIS IN BUTTER. 



PROTEOLYSIS IN BUTTER. 



It has long been known that butter differs from milk in its compo- 
sition onty in the relative amounts of the constituents present in the 
two. Among these constituents which early attracted attention as 
possible causes of storage flavor because of their chemical instability 
were the proteins, mainly casein. Proteins in the hydrated or moist 
condition in the presence of water are known to be unstable, and it is 
but natural that these substances, wherever they may occur in food, 
should be looked upon as possible sources of off flavor. It is almost 
certain that in butter containing no salt, or salt in an insufficient 
amount, or in butter that is not stored at sufficiently low tempera- 
ture, the proteins present do undergo hydrolysis and perhaps putre- 
faction and other obscure changes as weU. But the present work 
does not concern itself with such material. The problem is : If stor- 
age flavor develops in butter properly made and properly stored, do 
the proteins contribute in any way toward this off flavor ? 

Certain conditions in butter favor proteolytic changes, namely, the 
presence of water, bacteria, and of the proteolytic enzym known as 
galactase, which occurs i\ormally in milk. Other conditions, as low 
temperature, the presence of sodium chlorid, the partial inactivation 
of the galactase by pasteurization, tend to prevent or retard proteo- 
lytic changes. 

It has already been shown by other investigators that under con- 
ditions of comparatively high temperature and low salt the butter 
proteins will undergo changes. In the present work an attempt was 
made to determine whether the galactase present in butter made from 
pasteurized or from unpasteurized cream can digest casein in spite of 
the retarding influence of low temperature and high salt concen- 
tration. 

PREVIOUS WORK. 

Analytical difficulties. — When butter is melted and allowed to stand, 
the water present, containing the salt and casein in solution and in 
suspension, will settle to the bottom of the container, leaving the 
supernatant fat clear. The fat may be poured off and filtered if 
desired and at once used for quantitative work. However, aU of the 
fat can not be poured off, because part of it is thoroughly mixed with 
the particles of curd, so that after the most careful removal of fat 
by decantation a considerable amount is still left. Some of this 
residual fat can be removed by the addition of ether. This wiU dis- 
solve the fat on the upper surface of the curd solution and permit 
more fat to rise; but even three or four such washings with ether still 
leaves in the curd solution a considerable quantity of fat, probably 
20 grams of fat in 100 cubic centimeters of curd solution. It is 
obvious that such a mixture of fat, sodium chlorid solution, and 
66711°— Bull. 162—13 2 



10 CHANGE IN FLAVOR OF STORAGE BUTTER. 

casein suspension is not very well adapted to quantitative work. 
The material will not filter, nor can small samples of uniform com- 
position be easily withdrawn from it. 

In order to study the possible changes in the proteins of butter, 
this is the material to be used. In principle, the method of testing 
for the presence of active proteolytic enzyms in this material is no 
different from that used for other purposes, as, for instance, in tracing 
the proteolytic changes in ripening cheese or in animal tissue under- 
going autolysis. At first it would seem as if there should be no 
difficulty in making the usual nitrogen partition in this curd solution 
just as it is made on other viscous mixtures that are equally difficult 
to filter and sample. 

Evidently it was at the first step in the nitrogen partition that the 
difficulties began, for, to the best of our knowledge, none of the pre- 
vious investigators succeeded in precipitating the casein in the curd 
solution, filtering and determining nitrogen in the filtrate or the pre- 
cipitate, in such a manner as to enable the investigator to draw 
safe conclusions from the analytic data thus obtained. To this state- 
ment there are apparent exceptions. On adding acetic acid in usual 
amounts to some of the curd solution as if it were so much milk for 
the purpose of flocculating the casein and ffitering no flocculation is 
seen to occur and the mixture will filter so slowly as to make quanti- 
tative work unreliable for obvious reasons. If the curd solution be 
diluted with water until the casein can be flocculated by acetic acid 
in usual amounts, filtration is then rapid and the ffitrate can then be 
used for nitrogen determinations. The nitrogenous substances in 
butter, however, are about 75 per cent casein, so that on removal of 
the casein there is so little nitrogen left in the filtrate-from the diluted 
curd solution that the unavoidable errors in such work are very large 
when compared with the analytic data obtained. Still less certain 
are the results obtained on the nitrogen partition in such a filtrate, 
because the total nitrogen is too small for even that determination. 

In order to avoid the introduction of comparatively large errors, 
we made many attempts to increase the amount of curd solution used 
and to reduce the dilution before adding the precipitant. Acetic or 
other acids evidently were not previously used in quantities sufficient 
to flocculate the casein. Other precipitants, such as ferric chlorid, 
phosphotungstic acid, tannic acid, copper sulfate, etc., were tried. 
When added to curd solutions diluted with but two volumes of water, 
these precipitants will thoroughly flocculate tlie protein and give a 
filtrate that is clear, comes through rapidly, and can be used for 
quantitative work. Unfortunately, these precipitants remove from 
the curd solution practically all of the nitrogen, leaving too little in 
the filtrate. The water-soluble nitrogen in good butter is approxi- 
mately, one-fifth to one-tenth of the total, and in so far as the total 



PKOTEOLYSIS IN BUTTER 11 

nitrogen is represented by 1 per cent of curd, or about 0.1 to 0.2 per 
cent of nitrogen, it is necessary to use large amounts of curd solution 
for these precipitations in order that the filtrates may contain suffi- 
cient nitrogen for accurate determinations. 

Results hy Gray and others. — Several years ago (1906) Mr. C. E. 
Gray, then connected with the Dairy Division, began a study of the 
possible proteolytic changes in storage butter and their relation to the 
cliange in flavor. Following is his method of making the partition 
of nitrogen in butter: 

Total nitrogen. — Introduce 10 gi-ams of liutter into a Kjeldahl flask, digest, and 
distill as usual. 

To obtain nitrogen in other forms: Melt 2 kilos of butter in a hot-water jacketed 
funnel, temperature about 80° C. The melted butter is allowed to run into a cream 
separator with a special bowl having a capacity of 700 cubic centimeters without milk 
outlets. This was just large enough to hold all of the curd solution plus a small amount 
of fat. As the butter was fed in the bowl soon became filled and the excess of butter 
fat ran out, leaving the curd solution in the bowl. The larger part of the fat was 
washed out by feeding gasoline into the bowl. The last portion of gasoline was 
removed by feeding in water. The addition of water was stopped as soon as the out- 
flowing liquid carried particles of casein. In this way the curd solution in 2,000 gi-ams 
of butter was separated from most of the fat. The contents of the bowl were trans- 
ferred to a 1-liter flask, 25 cubic centimeters of 10 per cent ferric chlorid solution 
were added, and the total volume made up to the mark. The mixture was filtered on 
a 32-centimeter filter and the faintly colored filtrate used in the following determina- 
tions. Although but 600 to 700 cubic centimeters of filtrate were obtained, the 
results on aliquot portions were always calculated to 1,000 cubic centimeters. 

It is obvious that these filtrates contained only nitrogen not pre- 
cipitated by ferric chlorid; that is, nitrogen largely in the form of 
amino acids and ammonia. Certain peptones are precipitated by 
ferric chlorid.^ 

Total soluble nitrogen. — Transfer 50 cubic centimeter portions of the filtrate (cor- 
responding to 100 grams of butter) to Kjeldahl flasks and determine total nitrogen. 

"Amino and ammonia nitrogen." — Transfer a 200 cubic centimeter portion of the 
ferric chlorid filtrate to a 300 cubic centimeter volumetric flask. Add 1 gram of 
sodium chlorid and sufiicient 12 per cent tannic acid solution for maximal precipi- 
tation. Three or four cubic centimeters were usually required. Make up to the mark 
with distilled water, filter, and determine total nitrogen in 100 cubic centimeter 
portions of the filtrate, each of which corresponds to 133J grams of butter. 

"Ammonia nitrogen." — The method described by Van Slyke and Hart ^ was used. 

Transfer 100 cubic centimeters of the ferric-chlorid filtrate (corresponding to 200 
grams of butter) to a Kjeldahl flask, add 2 grams of magnesium oxid, and boil for about 
IJ hours, catching the distillate in N/20 acid. The excess of acid was titrated in the 
usual way. 

1 Siegfried, M. Zur Kenntniss der Phosphorfleischsaure. Zeitschriftfiir Physiologische Chemie, vol. 21,, 
no. 5/6, pp. 360-379. Strassburg, Apr. 2, 1896. 

Siegfried, M. Ueber Antipepton. Zeitschrift fiir Physiologische Chemie, vol. 27, no. 4/5, pp. 335-347. 
strassburg, June 24, 1889. See p. 342. 

2 Van Slyke, L. L., and Hart, E. B. Methods for the estimation of the proteolytic compoimds con- 
tained in cheese and milk. New York Agricultural Experiment Station, Bulletin 215, Geneva, September, 
1902. 



12 CHANGE IN FLAVOE OF STORAGE BTJTTER. 

Amino nitrogen is the difference between the sum of the amino and ammonia 
nitrogen and the ammonia nitrogen. 

Proteose and peptone nitrogen is the difference between the total soluble nitrogen 
and the sum of the amino and ammonia nitrogen. 

This method of studying the distribution of nitrogen in butter was 
used by Gray from 1906 to 1907 on a very large number of samples 
of butter churned from ripened, unripened, pasteurized, and unpas- 
teurized cream and stored at various temperatures. The plan of the 
investigation was very comprehensive. It aimed to ascertain the 
best conditions for the production of butter of best keeping quality 
and the chemical changes causing the off flavors of storage butter. 
This method was also used by us on one series of 24 samples in the 
spring of 1908. 

Gray's method of removing most of the fat from the butter by the 
use of the centrifuge was an improvement, without doubt. But for 
reasons to be made apparent presently the analytic data obtained 
by this method were not regarded as conclusive. More accurate 
data, it is believed, were later obtained by a method that is free from 
some of the objections that might be made to the method as originally 
devised by Gray. 

In Table 1 are some results obtained by Gray. The butter was 
obtamed from one lot of cream which was divided into eight portions 
from which eight separate churnings were made. The eight lots of 
butter were packed in 20-pound tubs and stored soon after churning, 
at —10° F. (—23° C). Analyses were made on the fresh butter, 
representing the condition of the nitrogen in the butter before storage. 
The two following series of analyses were made on the butter after 
different periods in storage. Two similar series of results were 
obtained by Gray on portions of the same lots of butter, stored at 
10° F. ( - 12° C.) and at 32° F. (0° C). The figures are not given 
here, but are in general similar to those in Table 1. 

From the results obtained by Gray it would seem that in the 
samples of butter examined slow proteolytic changes took place during 
storage. At least this is the inference to be drawn on the assump- 
tion that the method of obtaining the chemical data was free from 
avoidable errors. 



PROTEOLYSIS IN BUTTER. 



13 



Tabl^e 1. — Changes in the distribution of nitrogen in butter during cold storage 
(-10° F., -23° C.). 



Butter 
No. 


Age of 
sample. 


Total 
nitrogen. 


Total 

soluble 

nitrogen. 


Proteose 

and 
peptone 
nitrogen. 


Amino 
nitrogen. 


Aiumonia 
nitrogen. 


10.311 
10.312 
10.313 
10.314 
10.321 
10.322 
10.323 
10.324 


Days. 


206 
298 


206 
298 


206 
298 


206 
298 


206 
298 


206 
298 


206 
298 


206 
298 


Per cent. 
0.136 


Per cent. 
0. 0039 
.0070 
.0083 
.0041 
.0065 
.0074 
.0053 
.0067 
.0071 
.0045 
. 0061 
.0090 
.0020 
.0037 
.0073 
.0030 
.0053 
.0035 
.0037 
. 0047 
.0061 
.0065 
.0081 
.0087 


Per cent. 
0.0013 
.0032 
.0043 
.0026 
.0035 
.0026 
.0003 
.0032 
. 0041 
.0005 
.0023 
.0046 


Per cent. 
0.0019 
.0028 
.0030 
.0006 
. 0021 
.0039 
.0037 
. 0022 
.0017 
. 0028 
. 0025 
.0031 
.0018 
.0018 
.0014 
.0030 
.0036 
.0022 
.0029 
.0050 
.0030 
.0049 
.0048 
.0010 


Per cent. 
0. 00074 
. 00095 
. 00105 
. 00091 
. 00095 
. 00095 
. 00130 
.00134 
.00131 
.00115 
.00132 
.00128 
.00058 
.00066 
. 00070 
.00053 
.00061 
.00062 
. 00049 
. 00066 
. 00062 
.00087 
.00088 
. 00086 




.139 




.139 




.143 




.140 


.0014 
.0052 




.138 


.0021 
.0007 
.0003 




.139 




.0034 
.0007 
.0024 
.0068 


.141 







The method of Gray in a slightly modified form was used in the 
summer of 1908 and the spring of 1909. The results obtained were, 
in general, similar to those obtamed by Gray. They seemed to 
indicate that slow proteolysis was taking place. 

After using the method for a short tune several improvements 
suggested themselves. It will be noticed in Table 1 that the largest 
amount of nitrogen estimated in a ferric-chlorid filtrate (column 
headed "total soluble nitrogen") was equivalent to 0.009 per 
cent of nitrogen in the butter, or to 6.3 cubic centimeters N/10 
nitrogen. This is not a large amount. The largest difference between 
total soluble nitrogen before and after storage in Table 1 is that for 
sample 10.321, and is equivalent to not quite 4 cubic centimeters 
N/10 nitrogen. It seemed desirable so to change the method as to 
increase the amounts of nitrogen actually estimated. Whether 
proteolysis did or did not take place would then be decided with 
the aid of figures that are not so small that the unavoidable errors 
m such work are comparatively large. Filtration was so slow that 
evaporation undoubtedly took place to an unnecessarily large extent. 
The analyst could not be certam that 100 cubic centimeters of 
a ferric-chlorid filtrate obtamed after storage corresponded to ex- 
actly the same weight of butter as an equal volume of filtrate 
obtained before storage. Either filtration must be so rapid that 
evaporation may be disregarded because of its slight extent, or if 
filtration must be slow the volumes of filtrates should be care- 



14 CHANGE IN FLAVOR OF ST0EAC4E BUTTER. 

fully measured, so -that the weight of butter corresponding to any 
volume of filtrate can be definitely ascertained. 

Objections to ferric chlorid and tannic acid as protein precipitants. — 
Ferric chlorid as a protein precipitant was not wholly desirable 
because, as here used, it precipitated not alone the casein and other 
undigested proteins, but their immediate digestion products down 
to the peptone stage. The " f erric-chlorid filtrate" probably does 
not contain proteoses. The use of the term ''total soluble nitrogen" 
in this connection will lead to no confusion if it be borne in mind that 
it means nitrogen not precipitated by ferric chlorid. The figures 
for "proteose and peptone nitrogen" in Table 1 probably represent 
only some of the simpler peptones. The amount of nitrogen left in 
the ferric-chlorid filtrate is small, at least, when used for the separation 
of the different forms of nitrogen in butter-curd solution. 

Tannic acid as a protein precipitant is perhaps stiU more objection- 
able than ferric chlorid. It is well known that the precipitation 
limits of tamiic acid may be varied by the presence of salts, etc. 
Two results obtained with the aid of tannic acids are comparable 
only when the precipitant has been used in both cases under condi- 
tions that are exactly alike as regards the concentration of sodium 
chlorid, the amount of protein to be precipitated, the precipitating 
power of the samples of tannic acid used, etc.^ The greatest care 
must be taken in the use of the reagent to insure absolute uniformity 
in procedure. This is shown by the voluminous literature on the 
use of this reagent, in which the numerous difficulties and necessary 
modifications are pointed out. 

In addition to the difficulties just mentioned are those resulting 
from differences of opinion among investigators as to the best 
method of using the reagent. Van Slyke and Ilart,^ in their deter- 
minations of peptones in cheese are extremely careful to avoid an 
excess of tannic acid, probably because of the alleged solubility of 
the precipitate in excess of the precipitant. 

According to Bigelow and Cook ^ — 

* * * a considerable excess of tannin may be employed without any tendency of 
the reagent to dissolve the precipitate formed in excess. * * * 

Gray used tannic acid as directed by Van Slyke and Hart. 

It is not surprising that certain workers should advocate the dis- 
continuance of the use of tannic acid as a reagent for the determina- 
tion of amino acid nitrogen.* 

1 Bigelow, W. D., and Cook, F. C. The separation of proteoses and peptones from the simpler amino 
bodies. Journal of the American Chemical Society, vol. 28, no. 10, pp. 148&-1499. Easton, October, 1906. 

2 Loo. cit. 

3 Loc. cit., p. 1493. 

* Proceedings of the Twenty-sixth annual convention of the Association of Official Agricultural Chemists, 
United States Department of Agriculture, Biu-eau of Chemistry, Bulletin 132. Washington, 1910. See 

p. lot). 



PROTEOLYSIS IN BUTTER. 15 

The results obtained by Gray for amino nitrogen in Table 1 are not 
concordant, probably due to difficulties inherent in the use of tannic 
acid. Our own results are likewise difficult to interpret. In a certain 
series of analyses (butter No. 13.5) less amino nitrogen was founl in 
the butter after storage than before. The figures are not given here, 
as they are essentially similar to those of Gray. 

After obtaining a considerable number of results on the distribution 
of nitrogen in butter before and after storage, with the aid of ferric 
chlorid and tannic acid, we were unable to conclude that the results 
proved anything. It seemed more and more desirable to perfect a 
method that would permit the precipitation of the casein, then the 
estimation of proteoses by zinc sulfate, peptones by tannic acid, and 
ammonia by any of the methods that did not give too high results. 

In order to obtain a filtrate containing sufficient nitrogen for 
analytic work, the curd solution can not be diluted very much. In 
the undiluted condition it is a thick, viscous suspension of casein 
containing a variable amount of fat to which acetic acid may be added 
without any apparent effect. No flocculation can be observed. The 
mixture would filter extremely slowly. We made many attempts to 
find out why filtration was so slow. At first it was thought that fat 
particles clogged the filter paper. The first attempts were centered 
on the more thorough separation of the butter fat from the remainder 
of the butter, which in this paper is referred to as butter curd solution. 
It is obvious that unless the same amount of fat is removed in com- 
parative analyses of butter before and after storage, an error will 
be introduced because the fat remains in the precipitate, giving a 
smaller volume of more concentrated filtrate. The error from this 
source is probably much larger than anyone might suppose. The 
amount of fat present in the butter curd solution used for analytical 
purposes should be estimated so that corrections can be made if 
necessary. 

The butter fat may be separated from the remainder of the butter 
in more than one way. But obviously, when the curd solution and 
not the fat is wanted for quantitative work, the separation must con- 
sist of something more than a mere decantation of the melted fat. 
The butter fat and curd are so thoroughly mixed in the butter that 
when the butter is melted and allowed to stand, the separation between 
fat and curd solution is not complete. Most of the fat can be decanted, 
but very appreciable amounts still remain in the curd. Although 
Gray attempted to wash out the fat with the aid of gasoline, it is cer- 
tain that very much fat was still present in the material used for 
analysis. We tried to remove the fat with ether, but without success. 
The fat particles are embedded in curd and in this condition ether 
can not reach them. Besides, the ether could only be poured on top 
of the curd solution. Thorough mixing was inadvisable because of 



16 CHANGE IN FLAVOR OF STORAGE BUTTER. 

the possibility of forming emulsions that could not be separated. The 
use of ether was soon discontinued because of the possible dehydrating 
action of the ether upon the protein material and the subsequent 
obscuring of the results for nitrogen. The complete separation of fat 
from curd solution was then abandoned with the intention of esti- 
mating the amounts of fat present in portions used for analysis. In 
then- work on storage butter, Rahn, Brown, and Smith ^ did not record 
a quantitative separation of butter fat from curd solution. Instead, 
they proceeded as follows : 

Into a weighed 2-quart [fruit] jar about 500 grams of butter were poured and weighed 
to the 0.5 of a gi-am. Then 2 grams of hot water (70° C. to 75° C.) for every gram of 
butter was poured into the jar and the water and butter stirred occasionally for about 
an hour. The cover to which an arrangement for letting in air and drawing off water 
had been attached was put on and the jar inverted. After 15 or 20 minutes the 
water which had been separated from the fat was drawn off. Aliquot parts were 
taken for analysis. 

One objection to such a procedure lies in the fact that the water so 
separated from the fat contains too little nitrogen. The analytic data 
of Rahn, Brown, and Smith on nitrogen partition in butter are open 
to the same criticism as are Gray's data in Table 1. Their results 
differ little from those of Gray. Their work did not assist in explain- 
ing why acetic acid wiU flocculate casein in milk, but not in butter 
curd solution. This problem had been evaded by practically all 
who studied butter chemistry. 

In a recent investigation on the influence of preservatives on the 
keeping qualities and composition of butter and oleomargarin, 
Fischer and Gruenert - used the methods of Rahn, Brown, and Smith 
in their studies on proteolytic changes. They state that the addition 
of 3 per cent of salt to butter greatly retards, but does not entirely 
prevent, proteolytic and other changes in butter stored in a cool ceUar. 

NEW METHOD FOR DETECTING PROTEOLYSIS IN BUTTER. 

The influence of sodium chlorid on the preciintability of casein hy 
acetic acid. — Butter curd solution differs from milk in many respects; 
one of them is that butter curd solution may contam sodium chlorid 
m amounts rangmg from nothing up to saturation (over 30 per cent), 
depending upon the moisture and salt content of the butter. Per- 
haps the presence of the salt prevented flocculation of casein. 

During a previous investigation on the temperature of pasteuriza- 
tion for butter making ^ the followmg method of precipitating casein 

1 Loc. cit., p. 14. 

2 Fischer, K., and Gruenert, O. tJber den einfluss einiger Konservierungsmittel auf Haltbarkeit und 
Zusammensetzung von Butter und Margarine. Zeitschrift fiir Untersuchung der Nahrungs- und Genuss- 
mittel, vol. 22, no. 10, pp. 553-582, Berlin, Nov. 15, 1911. 

3 Rogers, L. A., Berg, W. N.,and Davis, Brooke J. The temperature of pasteurization for buttermaklng. 
United States Department of Agriculture, Bureau of Animal Industry, Circular 189, Washington, 1912. 
See p. 317. 



PROTEOLYSIS IN BUTTER. 17 

from buttermilk, milk, or skim milk was used. The object was to 
obtain a filtrate that was as concentrated in nitrogen as possible, and 
from which the casein had been quantitatively separated: 

Transfer 200 cubic centimeters of buttermilk to a 500 cubic centimeter volumetric 
flask. Add distilled water to about 450 cubic centimeters. Add one-fifth normal 
acetic acid (1.2 per cent) slowly until the casein separates completely in large flocculi 
leaving the supernatant liquid practically water-clear. In practically every case 
44 cubic centimeters of N/5 acetic acid was used and found sufficient for the purpose. 
After diluting to the mark and filtering, nitrogen determinations were made on the 
filtrate. 

The questions to be studied now were: Would the presence of salt 
in milk prevent the flocculation of the casein and the subsequent 
attempt at filtration ? 

In a sample of skim milk from which the casein can be easily floc- 
culated and filtered would the presence of added fat interfere with 
filtration ? 

Results were almost immediately obtained which threw a great 
deal of light on the difficulty. The following experiment is typical: 

Two hundred cubic centimeters of buttermilk obtained from a churning of pas- 
teurized cream were transferred to a 500 cubic centimeter volumetric flask. Water 
was added to about 400 cubic centimeters. On slowly adding 85 cubic centimeters 
N/10 acetic acid (0.6 per cent) the casein was completely flocculated. To a second 
500 cubic centimeter volumetric flask 200 cubic centimeters of the same sample of 
buttermilk was transferred. Thhty-six grams of sodium chlorid were added. This 
concentration of approximately 18 per cent is the concentration of salt in butter curd 
solution when the butter contains 16 per cent moisture and 3 per cent salt. Water 
was added, as before, to about 400 cubic centimeters. The addition of 85 cubic centi- 
meters N/10 acetic acid as before did not flocculate the casein. 

This experiment showed that the presence of salt very materially 
affected the precipitability of the casein. This was clearly a case 
where the physical condition of a colloid was so altered by the pres- 
ence of large amounts of electrolytes that it did not react toward a 
precipitant as it usually does. The acetic acid added certainly was 
not in excess. The slow addition of the acid without at any time 
resulting in even a partial precipitation of casein indicated that more 
acid was required. After adding 40 cubic centimeters more of N/10 
acetic acid, without any signs of flocculation, 10 per cent acetic acid 
(5/3 normal) was carefully added instead of the weaker N/10 acid. 
After adding 11 cubic centimeters of 10 per cent acetic acid, the 
casein was completely flocculated, and the mixture could be filtered 
rapidly, yielding a perfectly clear filtrate. 

In the presence of salt 300 cubic centimeters N/10 acetic acid were 
required for the precipitation of casein, which in the absence of salt 
would have been precipitated by 85 cubic centimeters of N/10 acetic 
acid. It is probable that observations of a somewhat simOar nature 
were simultaneously made by other investigators, as is evident from 
66711°— Bull. 162—13 3 



18 CHANGE IN FLAVOE OF STORAGE BUTTER. 

the following quotation from a paper by Schryver * which appeared a 
few months after the above-described experiment was made : 

During the course of some investigations on the action of formaldehyde on the pro- 
t.eins, the observation was made that this aldehyde, when added to an aqueous solution 
of Witte's peptone, produces a precipitate, and that the reaction could be either 
partially or completely inhibited by the presence of neutral salts. This phenomenon 
was also noticed some years ago by T. Sollman (American Journal of Physiology, 1902, 
vol. 7, p. 220). * * * 

Besides being extremely interesting theoretically, the observation 
just made on the effect of salt on the precipitation of caseiu 'was of 
interest practically because of its possible application to the separa- 
tion of casein from butter curd solution. The theoretical side of the 
phenomenon is discussed by Schryver in the above-mentioned paper. 

Method for the estimation of water-soluble nitrogen in butter. — After 
numerous experiments the following procedure was adopted for the 
separation of the fat from the bujter curd solution to be used for 
nitrogen determinations. As the method was new, many precautions 
were taken, some of which were later found to be unnecessary. This 
method of separating fat and casern from butter yields a filtrate that is 
well adapted, not alone to a study of the nitrogenous constituents of the 
filtrate, but to many other purposes as well. The filtrate, for example, 
contains the peroxidase which is practically always present in butter. 

To two or three large beakers, capacity 2 to 2| liters, transfer 5 to 6 
kilograms of the sample of butter to be studied. Two samples (one 
from raw, one from pasteurised cream, for example) may be worked 
with at one time. The larger the amount of butter taken the better. 
Place the beakers, properly marked, in a hot-air bath maintained at 
about 45° C. If the butter to be stored is packed in cans, five or six 
2-pound cans are placed in the hot-air bath to be melted. In the 
work here described the cans were taken out of the ice box late in the 
afternoon, allowed to warm up during the night at room temperature, 
and placed the next mornmg in the ah* bath, the temperature of 
which varied usually 2° above and below 45° C. The temperature 
should not be permitted to rise mxich beyond 45° C, because of the 
danger of coagulating some of the protem present. The high tem- 
perature at which Gray and Rahn, Brown and Smith (see pp. 11, 16) 
melted their samples of butter undoubtedly removed, by coagula- 
tion, comparatively large amounts of nitrogen from their filtrates. 

At this temperature from six to eight hours will be required for the 
complete melting of the butter and the settling of the curd solution. 
Stirring does little good. As fast as the butter fat forms a clear 
layer on top of the butter it may be poured off, care being taken that 
none of the curd solutionis lost at anytime. The loss of a few small 
particles of soHd protein is not material. This long heating at a tem- 

1 Sctuyver, S. B. Some investigations dealing with the state of aggregation of matter. Proceedings of 
the Royal Society, London, series B, vol. 83, no. B562, pp. 96-123. London, Dec. 19, 1910. See p. 96. 



PROTEOLYSIS IN BUTTER. 19 

perature that is perhaps best for proteolytic action is an objection to 
the method. It will be apparent, however, that the error introduced 
in this way is inappreciable. (See p. 20.) 

The melted butter fat is decanted until no more can be so removed 
without danger of losmg some of the curd solution. With the aid of a 
100 cubic centimeter pipette having its lower tip cut off to permit 
more rapid flow of viscous materials, remove the curd solution from 
the bottom of the beaker or can and transfer it to a dry, clean, 500 
cubic centimeter volumetric flask. Considerable fat will, of course 
be mixed with the curd solution, but by taking only part from the 
bottom of each vessel sufficient curd solution will be obtained from 
5 kilograms of butter to fill two 500 cubic centimeter flasks. Let 
stand till the next morning at room temperature. With the aid of a 
rapid-flow 100 cubic centimeter pipette, remove from the bottom of 
each of these flasks between 200 and 250 cubic centimeters of curd 
solution and transfer this to a clean, dry, 500 cubic centimeter volu- 
metric flask. From the original 5 kilograms of butter there have now 
been separated not quite 500 cubic centimeters of curd solution con- 
taining approximately 10 per cent of fat. The rest of the curd solu- 
tion containing much larger proportions of fat may be rejected. 

In a separate portion of the original sample of butter, determine 
moisture, curd, and salt. For this purpose the methods described in 
Bulletin 107 (revised edition). Bureau of Chemistry, page 123, were 
used. From these figures the weight of butter corresponding to a 
given volume of curd solution may be calculated if desired, but for 
the present work such calculation was not necessary. 

The curd solution should be well mixed, and then with the aid of a 
rapid-flow pipette a portion is transferred to a pycnometer (50 cubic 
centimeters capacity) and the weight ascertained. The object of this 
determination is to make certain that the curd solution used for 
analysis before and after storage of butter is practically the same so 
far as the density of the material is concerned. It is obvious that if 
the proportion of fat and salt solution differs very much in two sam- 
ples of curd solution obtained from the same lot of butter before arid 
after storage the specific gravity will be different. This determina- 
tion, when repeated on the same portion of curd solution, will show 
that it is possible to withdraw samples of uniform composition from 
the flask if care be taken to mix the contents well and to withdraw 
the sample rapidly before the fat rises to any appreciable extent. 

Portions of this curd solution may now be withdrawn for nitrogen 
determinations. To several clean, dry, 500 cubic centimeter volu- 
metric flasks transfer 100 cubic centimeter portions of curd solution, 
using a rapid-flow pipette and sampling quickly. This is the amount 
found most convenient when acetic acid is to be used as a precipitant. 
When other precipitants, such as ferric chlorid, are to be used, a larger 
volume may be taken. 



20 CHANGE IN FLAVOR OF STORAGE BUTTER. 

In our work we generally made three and sometimes four deter- 
minations on a single sample of butter, although two are perhaps 
sufficient. Each 100 cubic centimeter portion was used for one 
determination, so that as many such portions are to be pipetted into 
500 cubic centimeter flasks as determinations are wanted. (See 
Table 2.) In the remainder of the curd solution we determined fat 
by removing 10 cubic centimeter portions, transferring to absorbent 
paper, drying at the temperature of boiling water, and extracting the 
fat in a Soxhlet apparatus, as usual. These figures for fat are for 
the purpose of control only, and indicate how much of the volume 
of the portion of curd solution is taken up by fat. 

The undiluted curd solution may be allowed to remain in the 
stoppered flasks for 24 or 48 hours at room temperature without any 
apparent change in the results. In several cases even a week's 
standing was without effect. The concentration of salt is usually 
high enough to make the addition of other preservatives unnecessary. 
Under these conditions the proteolytic enzym present, galactase, is 
not very active; at least, on standing several days practically the 
same results are obtained as on freshly prepared curd solution. 

The curd solution is diluted to about 450 cubic centimeters with 
distilled water, mixed well and then 10 per cent acetic acid is added 
slowly, with constant mixing of the contents of the flask. It is 
obvious that sufficient acetic acid must be added to completely pre- 
cipitate the casein in the flocculent condition. This will occur 
generally when 18 to 22 cubic centimeters have been added. The 
curd solutions from pasteurized cream butter generally require the 
larger amount for flocculation. In Table 2 the amounts of acetic 
acid used are indicated. It seems that the more fat present the more 
slowly does the casein flocculate. On standing about 15 minutes the 
casein will be seen to have flocculated. The amount of acetic acid 
added should be recorded so that that same amount can be used after 
storage. In fact, all data should be recorded that might be necessary 
for the purpose of duplicating the determination exactly after storage. 
Dilute with distilled water to the mark and filter on a 32 centimeter 
folded filter (S. & S. No. 588 or 595) into a clean, dry, 500 cubic 
centimeter volumetric flask. The curd solutions should not be 
allowed to stand long after the addition of water. The filter stands, 
iclean funnels, and flasks were in readiness before the addition of water 
'and acid to the curd solutions, so that filtration was begun within a 
very few minutes. The entire contents of the flask were transferred 
to the filter paper. If the filtrate at first was cloudy, it was returned 
to the filter as often as necessary. Usually the first portions of 
filtrate, about 50 cubic centimeters each, were returned to the filter 
two or three times. In no case was a filtrate used for nitrogen deter- 
minations that was so cloudy as to indicate the presence of unpre- 
cipitated casein. Usually the filtration was begun in the afternoon 



PROTEOLYSIS IN BUTTER, 21 

and allowed to go on till the next morning. The funnel was covered 
with a well-fitting watch glass to minimize evaporation. 

On several occasions the precipitate on the filter paper was exam- 
ined at the end of the filtration for peptonizing bacteria. Such small 
numbers were found that their effect was inappreciable. It is 
obvious that the digestion of protein on the filter paper by bacteria 
or their enzymes might vitiate the results. We have no reason to 
believe that in any of the results appreciable errors were introduced 
in this way. 

The amount of acetic acid was varied a little when precipitating the 
casein from different portions of the same curd solution in order to 
find out whether, under the conditions of the work, small variations 
in the amount of acetic acid would give rise to undesirably large 
variations in the results. Of course, if insufficient acetic acid is 
added all of the casein will not be precipitated and the mixture wifi 
filter so very slowly that that alone will indicate incomplete pre- 
cipitation. The more completely the casein is precipitated the more 
rapid is the filtration. Slight excesses of acetic acid apparently have 
an inappreciably small solvent action on the precipitate. It is true 
that in precipitating casein from diluted milk an excess of even very 
dilute acetic acid is undesirable. In the presence of the sodium 
chlorid, however, conditions are so altered that the solvent action of 
the acetic acid is apparently very much diminished. In case of 
doubt, more rather than less acetic acid was used. 

The clear, slightly opalescent filtrate may be tested for complete- 
ness of precipitation by the addition of more acid or of alkali. In no 
case did the addition of small amounts of acid or of alkali (N/IO) to the 
filtrate result in the precipitation of protein. If sufficient alkali was 
added to make the filtrate alkaline to phenolphthalein, a precipitate 
was obtained which was probably calcium phosphate containing 
adsorbed protein. In appearance it resembled some protein pre- 
cipitates. The appearance of such a precipitate in an alkaline 
filtrate may, of course, be disregarded. Another way to test for 
completeness of precipitation is to use slightly different amounts of 
the precipitant. The results tabulated in Table 2 show that the 
amounts of acetic acid used were sufficient and that slight variations 
in the strength of the acid made no difference in the results. For the 
sake of certainty, the 10 per cent acetic acid solutions were titrated 
against standard alkali before being used as precipitants. This is 
especially desirable where the first determination is made in one 
laboratory and the second after cold storage in another. 

In a few instances ferric chlorid was used as a precipitant for the 
purpose of comparing the results with those obtained with acetic acid. 

Since evaporation can not be altogether prevented during the long 
filtration, it is necessary to be certain that equal volumes of filtrates 
obtained from the same lot of butter before and after storage corre- 



22 CHANGE IN FLAVOR OF STORAGE BUTTER. 

spond to exactly equal weights of butter, or, if through any con- 
siderable difference in evaporation the two filtrates are unequally 
concentrated, the difference in concentration must be ascertainable. 

After filtration is nearly complete — that is, after obtaining a little 
over 400 cubic centimeters of filtrate — its specific gravity is determined . 
A 50 cubic centimeter pycnometer was used. The same pycnometer 
filled a second time with some of the same filtrate will differ from its 
first weight by only 1 milligram. A little calculation "will show that 
before the nitrogen content of the filtrate can be appreciably varied 
through evaporation, the specific gravity will be varied so much more 
that its detection will be easy and require no fine weighmgs of the 
pycnometer. The pycnometer full of filtrate was always quickly 
.dried and weighed, and the weight recorded. The weight of a known 
volume of the clear filtrate is the best of the control figures, and 
together with the others should in every case show whether or not 
two filtrates of supposedly equal concentration really were so. The 
container in which the butter is stored might leak. There would 
result, not alone a loss in moisture, but in salt and nitrogen as well. 
Or if the container did not altogether prevent evaporation of water 
and a subsequent concentration of salt and nitrogen resulted, the 
pycnometer weighings will probably indicate the source of variation. 
When 100 cubic centimeters of curd solution were used, the filtrate 
contains so much sodium chlorid that considerable variation in 
specific gravity is possible. The amount of curd solution used corre- 
sponded to nearly 700 grams of butter. 

After weighing the pycnometer full of the clear filtrate, 400 cubic 
centimeters of it were transferred to a Kjeldahl flask and total 
nitrogen was determined in the usual way. The remainder of the 
filtrate was measured and the total volume of filtrate obtained was 
recorded. If filtration was very slow, sometimes less than 400 cubic 
centimeters were used. The results were calculated to 400 cubic 
centimeters. The weight of the precipitate and mclosed filtrate was 
ascertained to the nearest gram on a torsion balance, and recorded. 
The 400 cubic centimeters of filtrate actually kjeldahled and titrated 
corresponded to 560 grams of butter. Rahn, Brown, and Smith ^ 
determined nitrogen in butter not precipitated by acetic acid. They 
• washed butter with water in the proportion of 1 gram of butter to 
2 cubic centimeters wash water, transferred 100 cubic centimeters 
of these washings to a flask, added acetic acid, filtered off the pre- 
cipitated protein, and determined total nitrogen in 25 cubic centimeters 
of filtrate, which corresponded to not quite 12^ grams of butter. 
Apparently they obtained very few results with acetic acid and did 
not use them in drawing their conclusions. It was pointed out 
before (see p. 10) that when butter curd solution is diluted with 
sufficient water it may be treated with acetic acid as if it were so 

1 Loc. cit., pp. 14-15. 



PEOTEOLYSIS IN BUTTER. 23 

much diluted milk and the casein will be flocculated, permitting very 
satisfactory filtration. The filtrate, however, contains an unde- 
sirably small amount of nitrogen. 

I'he figure for total nitrogen in the filtrate taken for analysis was 
multiplied by 5/4, and the result recorded as the number of cubic 
centimeters of N/5 nitrogen in 100 cubic centimeters butter curd solu- 
tion. (See Table 2.) From the data it is obvious that the unavoid- 
able errors of ordinary nitrogen determinations are very small when 
compared with the amount of nitrogen determined, 

A separation of the various forms of nitrogen in the filtrate from 
the casein precipitation could easily have been made. But it was 
considered desii-able first to find out whether this filtrate contamed 
any more nitrogen after storage than before. If it did, indicating 
that proteolysis was taking place, then a more detailed examination 
of the filtrate would have been made. But the filtrates differed very 
little in their total nitrogen content before and after storage, indicating 
that proteolytic changes were not taking place to any great extent, 
and other lines of work were begun. 

The above-described method for the estimation of water-soluble 
nitrogen in butter was used in the summer of 1910 and in the spring 
of 1911. (See Table 2.) 

Description of samples. — Samples Nos. 42 and 40 were churned from 
the same lot of sweet cream, half of which was churned unpasteurized 
(butter No. 42) and half of which was churned after pasteurization 
(butter No. 40). Samples 52 and 50, and samples 65 and 62 were 
likewise obtained from churnings of two lots of sweet cream, part of 
which was pasteurized, part of which was not, before churning. 
(See Table 2.) The expectation was that if galactase is active 
in butter durmg cold storage, the figures for water-soluble nitrogen 
in samples 42, 52, and 65 would become larger, for in these sam- 
ples of butter from unpasteurized cream the conditions for pro- 
teolysis were as favorable as they ordinarily can be. No great 
changes were expected in the water-soluble nitrogen in the controls 
Nos. 40, 50, and 62 because at the temperature of pasteurization 
used, 75° C. (167° F.) in a ''flash" pasteurizer, the galactase ordi- 
narily present in butter is strongly inactivated or partly destroyed. 

Samples 51 1 and 523 were churned from the same lot of pasteurized 
cream. Sample No. 511 contained a proteolytic enzym preparation 
obtained from cultures of acid-forming bacteria which also liquefied 
gelatin. Twelve grams of dry enzym powder were worked into 
about 30 pounds of butter with the salt. The control lot of butter 
No. 523 was made in the same way, except that a similar amount of 
enzym preparation was added after it was first boiled in water. 
Similarly, sample No. 466 was churned from pasteurized cream and 
contained an added proteolytic enzym preparation, while its con- 
trol. No. 478, contained an equal amount of the enzym that had 



24 



CHANGE IN FLAVOR OP STORAGE BUTTER. 



been boiled before being worked into the butter. The object of study- 
ing these samples was to ascertain whether the proteolytic enzym 
secreted by bacteria often present in cream can digest any of the 
butter proteins, under storage conditions. 

All of the samples in Table 2 were churned in the experimental 
creamery in Albert Lea, Minn., in the summer of 1910. They 
were stored a short time in the creamery cooler and then shipped 
to cold storage at 10° F. (minus 12° C.) in Chicago. Naturally, care 
was taken to move the material into cold storage as soon after churn- 
ing as possible. When samples were shipped from Chicago to Wash- 
ington for analysis, they were removed from the railroad station as 
soon after their arrival as possible and placed in a refrigerator in the 
laboratory maintained at a few degrees below 0° C. 

At appropriate times samples of the butter were sent to competent 
judges for scoring. The scores are indicated in their appropriate 
places in Table 2. 

Table 2. — Analytic data and scores — Water-soluble nitrogen ^ in sweet-cream butter be/ore 
and ajter storage. 10° F. {-12° C). 







N/5 water-soluble 


nitro- 
















gen in 100 c. c. 


butter 




Butter 


scores. 


Volume 


Precipi- 


Treatment of cream. 


Butter 
sample 


curd solution. 




Age of 
sample. 






of butter 

curd 
solution 
used for 
analysis. 


tant, 
10 per 
cent 
acetic 
acid. 




No. 


Before 


After 


Differ- 


Before 


After 






storage. 


storage. 


ence. 




storage. 


storage. 






C.c. 


C.c. 


C.c. 


Days. 






C.c. 


C.c. 






( 32.8 


28.9 


- 3.9 








f 50 


7 


Unpasteurized 


42 


34.0 


28.7 


- 5.3 


■ 265 


85 


85 


\ 50 


7 






19.9 


14.0 


- 5.9 








100 


(=) 






30.3 


31.9 


1.6 








( 100 


10 




52 


{ 28.9 


30.3 


1.4 


■ 251 


87 


85 


i 100 


14 






1 28.7 


29.0 


0.3 








[ 100 


16 






f 26.1 


21.2 


- 4.9 








( 100 


16 




65 


25.1 


21.2 


- 3.9 


250 


90 




\ 100 


20 






t 26.0 


21.2 


- 4.8 








{ 100 


22 






( 23.4 


24.6 


1.2 








( 60 


7 


Pasteurized 


40 


\ 23.4 
13.4 


23.6 
16.0 


0.2 
2.6 


205 


91 


90 


\ 50 
t 100 


7 




(') 






19.4 


20.7 


1.3 








J 100 


16 




50 


\ 19.0 


19.0 


0.0 


• 251 


93 


91 


i 100 


18 






18.7 


19.4 


0.7 








1 100 


20 






22.8 


17.6 


- 5.2 








( 100 


16 




62 


\ 22.3 


17.6 


- 4.7 


. 250 


92 




\ 100 


20 






t 22.7 


16.8 


- 5.9 








( 100 


22 


Pasteurized. Dry pro- 


1 


31.8 


26.6 


- 5.2 








1 100 


18 


teolytic enzym 


[ 511 


I 32.6 


26.5 


- 6.1 


• 250 


92 


92 


\ 100 


20 


added s 


J 


35.1 
f < 32. 7 


27.1 
72.9 


- 8.0 
40.2 








1 100 

(5) 


12 




(.') 




466 


\ < 86. 1 


86.6 


0.5 


296 


92J 


93 


1 (') 


9 






m7.9 


84.1 


-33. 8 








(') 


5 


Pateurized. Heated 


1 


22.4 


23.5 


1.1 








( 100 


18 


bacterial proteolytic 


\ 523 


\ 22.8 


23.5 


0.7 


■ 250 


93 


93 


\ 100 


20 


enzym added 


J 


I 23.6 
f < 22. 


24.4 


0.8 








1 100 


12 




478 


<23.9 
* 75. 


"'55.' 6" 

68.7 


"'si.'i' 

- 6.3 


■ 296 


93J 


93i 


1 e) 

1 (') 


'-' 9 






m2.8 


72.3 


-40.5 








(') 


5 



1 Calculated total nitrogen in 100 cc butter curd solution equivalent to nearly 700 grams butter=350ccN/6 
nitrogen. 

2 Eight c. c. 10 per cent ferric chlorid solution. 

3 Analytical work, June, 1911, on butter, buttermilk, etc., by R. P. Norton. 
* N/5 water-soluble nitrogen in 1,000 grama butter. 

s Equivalent of 800 grams of butter. 

6 Ten e. c. 10 per cent ferric chlorid solution. 

' Equivalent of 400 grams of butter. 



PROTEOLYSIS IN BUTTER. . 25 

Discussion of results, Tahle 2. — In general, fresh butter made from 
unpasteurized cream (No. 42, for example) has a little more water 
soluble nitrogen than the corresponding sample (No. 40) churned 
from some of the same lot of cream after pasteurization. Butter- 
milk from raw-cream butter contains more water-soluble nitrogen 
than the corresponding sample of buttermilk from pasteurized 
cream. (Compare samples 13 and 14, Table 3.) In sterilized skim 
milk the soluble nitrogen content is still lower. (Compare samples 
20, 22, and 24 with 14, 16, and 18, Tables 3 and 4.) These differences 
between pasteurized and unpasteurized samples are very likely due 
to the partial or entire coagulation of the milk albumin, and its 
removal from the water-soluble condition. 

The coagulation of water-soluble nitrogenous substances in butter- 
milk due to high pasteurizing temperatures was shown in a previous 
publication from the Dairy Division laboratories.^ 

It is believed that samples 42, 52, and 65 contained more water- 
soluble nitrogen than their controls Nos. 40, 50, and 62, because 
there was a partial precipitation of protein during the pasteuriza- 
tion of the cream from which the latter were made and not because 
the galactase undoubtedly present in Nos. 42, 52, and 65 was active. 
If it were, there should have been an increase in the amount of 
water-soluble nitrogen after storage. In so far as there was none, 
it is inferred that the activity of the galactase was inhibited by the 
combined effect of the salt and cold storage. 

The differences between the amounts of water-soluble nitrogen in 
the different samples of butter before and after storage are not very 
large, except in samples 466 and 478, and they represent in all 
probability the unavoidable error in such work. It is to be borne 
in mind that the first analysis was made in Albert Lea, Minn., and 
the second on a different lot of cans m Washington, D. C. Under 
such circumstances the differences are not considered large. In 
samples 466 and 478 it is probable that the figures obtained for 
nitrogen are erroneous. 

CONCLUSIONS. 

From the data obtained it is evident that proteolysis did not take 
place to any appreciable extent in the samples studied. Nor was 
there any simple or obvious relation between the figures for nitrogen 
and the butter scores. 

As the following calculations show, the method for detecting 
proteolytic action in butter is quite delicate and should lead to the 
detection of proteolysis were it appreciable. In a determination of 
water-soluble nitrogen in butter 100 cubic centimeters of curd solu- 
tion were used. This is equivalent to a little over 700 grams of butter 

1 Rogers, L. A., Berg, W. N., and Davis, Brooke J. Loc. cit., p. 317. 
66711°— Bull. 162—13 4 



26 CHANGE IN FLAVOR OF STORAGE BUTTER. 

containing 13 per cent of moisture. A nitrogen determination was 
made in 400 cubic centimeters of the filtrate from the acetic acid 
precipitation, equivalent to 560 grams of butter. The butter used 
contained an average of 0.9 per cent of curd. The 700 grams of 
butter contained, therefore, 700x0.009x0.1567 = 0.9872 grams of 
nitrogen, equivalent to 352 cubic centimeters N/5 nitrogen. 

Suppose that during the cold-storage period 5 per cent of the casein 
was slightly proteolyzed and became water soluble. It should be 
borne in mind that the method of detecting proteolytic action here 
described will detect it in its first stages. In this respect the method 
possesses undoubted advantages over others in which results are 
obtained for variations in amounts of proteoses, amino acids or 
ammonia, which correspond to later and later stages in the digestive 
process. It is here supposed that the first step in the digestion of 
5 per cent of the casein has taken place, the rest of the protein 
remaining unchanged. Allowing for 30 cubic centimeters N/5 nitro- 
gen already present in the 100 cubic centimeters of curd solution 
as water-soluble nitrogen, there would be formed by the proteolysis 
0.05 X 320 cc= 16 cubic centimeters N/5 nitrogen in the water soluble 
form in addition to the 30 cubic centimeters originally present. The 
titrations made after storage would then show 16x4/5 or 12 cubic 
centimeters more of N/5 nitrogen than before storage. From the 
results obtained it is probable that this increase, had it taken place, 
would have been detected. 

A protein, such as casein, can of course undergo more than one 
kind of chemical change. These changes may be hydrolytic, oxida- 
tive, or putrefactive. It is obvious that the methods used in this 
work would detect the hydrolytic change only. Some work on the 
possibility of oxidative changes in the protein in butter is described 
on page 64 et seq. 

PROTEOLYSIS IN MILK. 

POSSIBLE OBJECTION TO THE NEW METHOD FOR DETECTING PRO- 
TEOLYSIS IN BUTTER. 

An apparent objection to the method of studying possible pro- 
teolytic changes in butter just described lies in the fact that a 
long time (five days) may elapse between the beginning and end 
of the determinations of nitrogen, during which time the galactase 
or bacterial proteolytic enzyms present in butter may be active. 
The results would not represent proteolysis during cold storage but 
proteolysis during the determinations of nitrogen. For this reason 
the following experiments were made. 

The objects of these were two-fold: First, to ascertain by a method 
that was free from the objections to the method for butter whether 



PKOTEOLYSIS IN MILK. 27 

galactase could digest protein in an 18 per cent sodium chlorid 
solution. Second, whether the sodium chlorid usually present in 
butter curd solutions can inhibit the proteolytic action, not alone of 
the small quantity of galactase ordinarily present in butter, but also 
the action of larger amounts of proteolytic enzyms that might find 
their way into butter m any one of several ways, as, for instance, 
the proteolytic bacteria that may grow in the milk and cream. 

THE INHIBITING EFFECT OF SODIUM CHLORID AND COLD STORAGE UPON 
THE ACTIVITY OF GALACTASE IN BUTTERMILK. 

Six lots -of buttermilk of about eight liters each were obtained 
from three churnings of unpasteurized sweet cream and from three 
churnings of pasteurized sweet cream. In Table 3, page 29, is indi- 
cated the number of the lot of butter corresponding to each lot of 
buttermilk. Buttermilk samples 13 and 14 were obtained from the 
churnmg of one lot of sweet cream, half of which was churned un- 
pasteurized (butter No. 42, buttermilk No. 13) and half of which 
was pasteurized before churning (butter No. 40, buttermilk No. 14). 

To each liter of buttermilk 5 cubic centimeters of chloroform and 
180 grams of sodium chlorid were added. These samples were 
intended to represent approximately butter minus butterfat. In 
such material a study of possible proteolytic action could be made 
in a comparatively short time, since no time is necessary for the 
melting of the butter or the separation of the fat. 

The buttermilk was then sealed in cans, each containmg 600 cubic 
centimeters of the sample. These cans, which were also used for 
butter, were of heavy tin, thoroughly lacquered. The smaller part 
of these samples remained in the creamery cooler; the rest were 
shipped to cold storage in Chicago. The samples were not removed 
from the cooler for analysis until it was certain that sufficient time 
had passed to permit the transportation, by refrigerator freight, of 
the samples from Albert Lea, Minn., to Chicago, and the placing of 
the samples in the cold-storage rooms. It was intended that the 
first analysis of these samples should show, as closely as possible, 
the amount of water-soluble nitrogen present m the material as it 
went into cold storage, and not as it left the churn. It is highly 
probable that galactase, even in the presence of 18 per cent sodium 
chlorid, can slowly digest protem material, if the temperature is 
above that of cold storage as it is ordinarily practiced. It seems 
reasonable to suppose that the proteolysis in butter observed by 
Rahn, Brown, and Smith ^ was due, in part at least, to the compara- 
tively high temperatures at which their butter was stored. But in 
so far as this investigation is concerned with possible chemical 

> Loc. cit. 



28 CHANGE IN FLAVOR OF STORAGE BUTTER. 

changes that may take place in butter while in cold storage and not 
at higher temperatures, the times at which analyses were made were 
always chosen so as to give results as nearly representative of the 
condition of the material immediately before and after cold storage 
as possible. 

Method of measuring the activity of galactase in huttermilk. — When 
it was reasonably certam that the other samples of buttermilk had 
reached the cold storage, samples were removed from the creamery 
cooler and water-soluble nitrogen was determined in them by prac- 
tically the same method as was used for butter. The buttermilk 
was treated as if it were so much butter curd solution entirely freed 
from fat. All the precautions that were taken in the work on butter 
were taken here also. The method is described on page 18. 

The first analyses were made in September and October, 1910. 
For the analyses made in December, 1910, and m June, 1911, samples 
were shipped from cold storage in Chicage by express to Washington. 
Upon their arrival the material was at once transferred to the re- 
frigerator in the Dairy Division laboratories, where it remained till 
used for analysis. The total time durmg which the samples were 
out of cold storage was as short as possible. 

Results. — From the results obtained on samples 13, 15, and 17, 
summarized in Table 3, it is evident that in buttermill^; obtained 
from raw-cream butter the activity of galactase is practically en- 
tirely inhibited by the presence of 18 per cent of sodium chlorid and 
by the low temperature, 0° F. ( — 18° C.) of the cold storage. When 
some of this same material is allowed to remain for a long time at 
room temperature, the galactase apparently becomes much more 
active, because the casein is seen to clot and the mixture assumes the 
appearance of one in which digestion is going on. 

Samples 14, 16, and 18 were to serve as controls on those of the 
other three. Although galactase in cream is not entirely destroyed 
by ordinary pasteurization, it is partly inactivated.' It was expected 
that the figures for water-soluble nitrogen in samples 14, 16, and 18 
would change little during storage, thereby affording a check on the 
correctness of the work. The figures for water-soluble nitrogen in 
samples 13, 15, and 17, had they increased during storage, could 
then have been considered as obtained by a method that showed no 
change where none is to be expected. 

' Rogers, L. A., Berg, W. N., and Davis, Brooke J. Loc. cit., p. 318. 



PROTEOLYSIS IN MILK, 



29 



Table 3. — Effect oj sodium chlorid and cold storage (0° F.,—18° C.) upon the activity oj 

galactose in buttermilk. 



Buttermilk obtained from churnings of unpas- 
teurized sweet-cream buttei. 


Buttermilk obtained from churnings of pas- 
teurized sweet-cream butter. 


Butter- 
milk, 
lot No. 
16.612, 
sample 
No. 


Butter, 
lot No. 
10.311, 
sample 
No. 


N/5 water-soluble 

nitrogen in 100 c. e. 

buttermilk. 


10 per cent 
acetic acid 
used as pre- 
cipitant for 

200 c. c. 
buttermilk. 


Butter- 
milk, 

lot No. 
10.622, 

sample 
No. 


Butter, 
lot No. 
10.321, 
sample 
No. 


N/5 water-soluble 

mtrogeninlOOc.c. 

buttermilk. 


10 per cent 
acetic acid 
used as pre- 
cipitant for 


Age. 


N/5N. 


Age. 


N/5 N. 


200 c.c. 
buttermilk. 


13 
15 
17 


42 

52 
65 


Days. 
25 

116 

301 

10 
124 
297 

10 
101 
280 


C.c. 
39.2 
36.8 
40.7 
41.0 
37.2 
37.4 
42.1 
40.9 
43.4 
42.6 
39.5 
40.0 
42.6 
45.3 
45.1 
46.3 
41.1 
38.7 


C.c. 
20 
18 
20 
18 
20 
18 
16 
20 
16 
20 
16 
20 
14 
12 
14 
12 
14 
12 


14 
16 

18 


40 
£0 

62 


Days. 
25 

116 
301 

10 
124 
297 

10 
101 
286 


C.c. 
36.1 
38.2 
39.1 
36.3 
38.3 
31.1 
30.3 
31.9 
30.3 
30.1 
28.6 
35.0 
36.3 
35.2 
36.3 
33.6 
34.8 


C.c. 
18 
12 
12 
18 
12 
20 
24 
20 
24 
20 
24 
22 
18 
22 
18 
22 
18 



179 cubic centimeters N/5 nitrogen=average total nitrogen in 100 cubic centimeters buttermilk. 

The results on these samples of buttermilk confirm the results 
obtained on the corresponding samples of butter. It is practically 
certam, for example, that butter sample No. 42 and the buttermilk 
obtained from it both contained galactase, though in different 
amounts. Under the conditions of the experiments proteolytic action 
was uniformly inhibited, both in the butter and in the buttermilk. 

As the following calculations show, the method used for the detec- 
tion of proteolytic action in buttermilk (skim milk) is quite delicate 
and should lead to the detection of proteolytic action were it appre- 
ciable. In the determination of water-soluble nitrogen in buttermilk 
200 cubic centimeters were taken, which contained very close to 360 
cubic centimeters N/5 total nitrogen. Let it be assumed that during 
the cold storage period 5 per cent of the casein became water soluble. 
Allowing an average of 80 cubic centimeters N/5 nitrogen in water- 
soluble form originally present in the 200 cubic centimeters of butter- 
milk, there would be formed by the proteolysis 0.05X280 = 14 cubic 
centimeters N/5 water-soluble nitrogen. In the actual determination 
200 cubic centimeters of buttermilk were diluted to 500 cubic centi 
meters precipitated with acetic acid, and two 200 cubic centimeter 
portions of the filtrate were used for nitrogen determinations. There- 
fore if 5 per cent of the casein had become hydrolized the titrations 
after cold storage would have been 5.6 cubic centimeters higher than 
the corresponding titrations before storage. It is probable that this 
increase would have been detected. 



30 



CHANGE IN FLAVOR OF STORAGE BUTTER. 



THE INHIBITING EFFECT OF SODIUM CHLORID AND COLD STORAGE 
UPON THE ACTIVITIES OF PROTEOLYTIC ENZYMS IN STERILIZED 
SKIM MILK. 

Description of sam'ples. — Several 5-liter flasks full of skim milk were 
kept in a steam sterilizer for about two hours at a temperature vary- 
ing between 94° and 99° C. 

Three 3-hter portions were measured out roughly and rapidly while 
the skim milk was hot, into bottles, samples No. 20, 22, and 24. A 
weighed quantity of the enzym preparation was then added. The 
amounts are given in Table 4, page 30. 

After cooling, 540 grams of sodium chlorid was added to each 
sample. The bacterial enzym was prepared from cultures of an acid- 
forming bacterium that secreted a proteolytic enzym. The usual 
method of precipitating with alcohol, etc., was used. The dry en- 
zym preparation was tested before it was used in the experiments 
and found to be proteolytically active. The other enzym prepara- 
tions were the ordinary commercial ones. 

Three-liter portions of the skim milk were quickly cooled to 35° C. 
To each was added 540 grams of sodium chlorid, to which there had 
been previously added the amount of enzym indicated in the table. 
Obviously, samples 20, 22, and 24 were controls on Nos. 19, 21, and 23. 

These samples were canned as before. Most of these were sliipped 
to cold storage in Chicago, where they were maintained at a tempera- 
ture of 20° F. (— 7° C). Determinations of water-soluble nitrogen 
were made by the method already described in a previous publica- 
tion ' and in this paper page 18. 

Table 4. — Effect oj sodium chlorid and cold storage upon the activities oj proteolytic 
enzymes in sterilized shim milh stored at 20° F. ( — 7° C). 



Skim 
milk, 
lot No. 
10.612, 
sample 
No. 


Composition of 
mixtures. 


N/5 water- 
soluble ni- 
trogen 2 in 
100 c.c. skim 
milk after 
75 days' 
storage. 


10 per cent 
acetic acid 
used as pre- 
cipitant for 
200 c.c. skim 
milk. 


Skim 
milk, 
lot No. 
10.022, 
sample 
No. 


Composition of 

control 

mixtures. 


N/5 water- 
soluble ni- 
trogen 2 in 
100 c.c. skim 
milk after 
77 days' 
storage. 


10 percent 
acetic acid 
used as pre- 
cipitant for 
200 c.c. skim 
milk. 


19 


Skim milk, 3 liters 
Dairy salt, 540 

grams. 
Bacterial 


C.c. 
44.9 
48.1 


C. c. 
20 
10 


20 
22 
24 


Skim milk, 3 liters 
Dairy salt, 540 

grams. 
Bacterial 


C.c. 
28.1 
33.1 


C.c. 

20 
10 




Enzym (dry), 15 

grams. 
Skim milk, 3 liter.s 
Dairy salt, 540 

grams. 
Pancreatin, 3 

grams, dry (U. 

g. P.). 
Skim milk, 3 liters 
Dairy salt, 540 

grams. 
Pepsin(U.S. P.), 

dry, 3 grams. 






Enzym (boiled), 

15 grams. 
Skim milk, 3 liters 
Dairy salt, 540 

grams. 






21 


118.2 
121.0 


20 
10 


22.8 
28.5 


20 
10 


23 


54.1 
65.5 


20 
10 


P.), boiled, 3 

grams. 
Skim milk, 3 liters 
Dairy salt, 540 

grams. 
Pepsin (U.S. P.), 

boiled, 3 grams. 


19.8 
24.2 


20 
10 













1 Rogers, L. A., Berg, W. N., and Davis, Brooke J. Loc. cit., p. 315. 

2 198 c. c. N/5 nitrogen=average total nitrogen in 100 c. e. skim milk. 



PEOTEOLYBIS IN MILK. 31 

Results. — In sample No. 19 there was present a large quantity of 
a proteolytic enzym of bacterial origin that was known to be active 
on gelatin. It is highly probable that much more enzym was present 
here than there is ever present in butter, and the figures indicate 
plainly that the salt strongly inhibited its action durmg the period 
under observation. It is, of course, possible that, given a period of 
time greatly exceeding ordinary storage periods, further proteolysis 
in this sample might have been observed. 

In samples 21 and 23 digestion took place to a large extent. In 
No. 21 approximately two-thirds of the total protein had become 
water soluble. 

On samples 20, 22, and 24 practically identical results were ob- 
tamed before and after storage, as would be expected. This indi- 
cated that in the controls no proteolytic changes were detected. 

CONCLUSIONS. 

It is evident from these results that in the presence of very large 
amounts of strongly active proteolytic enzyms, proteins will be 
hydrolized even in cold storage (or in transit) and in strong salt solu- 
tions. But there is no reason to suppose that at any time such 
amounts of enzyms are ever found in butter. 

It is very probable that samples 21 and 23 contained several thou- 
sand times as much proteolytic enzym as is present in ordinary 
butter of any kind. 

However, it must be borne in mind that the claim is not made 
that sodium chlorid does not exert an inliibiting influence on pro- 
teolytic action. Whether it does or does not depends upon the 
conditions of the experiment. It is comparatively easy to show 
that the action of pepsin-acid can be inliibited very strongly by 
large amounts of sodium chlorid. In the spring of 1909 some experiT 
ments were made in which the speed of digestion of casein in several 
pepsin-acid solutions was compared with that in the same solutions 
to which 20 grams of sodium chlorid to 100 cubic centimeters of acid 
solution had been added. The presence of the salt almost completely 
inliibited the action of the pepsin-acid during the time of the experi- 
ment, 40 minutes' digestion, but it is, of course, possible that proteo- 
lytic action would have taken place had the digestion period been 
several months. The method of comparing speeds of digestion was 
that described by Gies.^ In general, the statement that sodium 
chlorid does inhibit proteolysis is true, therefore, at low tempera- 
tures, when the amount of enzym is small and the digestion period 
(storage period) is long or when the amount of enzym is large and 
the digestion period is short, as in ordinary digestion experiments. 

1 Berg, William N., and Gies, William J. Studies of the effects of ions on catalysis, with particular 
reference to peptolysis and tryptolysis. Journal of Biological Chemistry, vol. 2, no. 6, pp. 48&-646. New 
York, March, 1907. 



32 CHAKGE IN FLAVOE OP STOEAGE BUTTER. 

The statement that sodium chlorid does not inhibit proteolytic 
action is true at comparatively high temperatures when the amount 
of enzym is very large and the digestion period very long. The 
apparently contradictory statements are the results of testing the 
action of the sodium chlorid over a wide range of enzym concentra- 
tion and over widely varying digestion periods. 

Several investigators have studied the action of sodium chlorid on 
the tryptic digestion of casein. Their conflicting statements are, of 
course, easily accounted for by the fact that the experiments were 
made under conditions that were not uniform with regard to the 
concentration of sodium chlorid, the relative proportions of alkali, 
trypsin, and casein, etc. Their work is summarized by Robertson.^ 

THE INDIRECT ACTION OF BACTERIA. 

The improbability that the proteolytic enzyms are responsible for 
the difference in keeping quaUt}'^ between unpasteurized and pasteur- 
ized sweet-cream butter is shown by the work given in detail in the 
preceding pages. The bacteria are another possible factor removed 
by pasteurization. WliUe it is certain that they do not grow at the 
low temperature of commercial storage, it is possible that the pres- 
ence of a large number of living cells, or various active enzyms 
which may possibly be liberated by the death of the bacteria, may 
have an influence on the flavor of the butter. If the bacteria 
destroyed by the pasteurization could be replaced in the cream their 
influence on the flavor should be shown in a comparison of the butter 
made from this reinoculated cream and that made from a part of the 
same pasteurized cream, but without the addition of bacteria. 
Before this could be done intelligently it was necessary to obtain a 
general knowledge of the bacteriological content of the raw cream. 
The normal bacteria of the cream from one skimming station was deter- 
mined by sampling every day and plating on lactose agar. After 7 
days' incubation at about 30° C. the plates were counted and all of 
the colonies on a representative plate were transferred to litmus milk 
tubes. These were incubated and examined after 2, 5, and 14 days. 
This enabled a separation into high acid forms which curdled the milk 
in less than 2 days; low acid forms forming acid but curdling the 
milk slowly or not at all; alkali formers, peptonizers, and those that 
produce no visible change in the milk. These groups were calculated 
as percentages of the total bacteria, and the results are given in 
Table 5. 

2 Robertson, T. Brailsford. On some chemical properties of casein and their possible relation to the 
chemical behavior of other protein bodies, with especial reference to hydrolysis of casein by trypsin. 
Journal of Biological Chemistry, vol. 2, no. 4, pp. 317-383. New York, January, 1907. See p. 355. 



THE INDIRECT ACTION OF BACTERIA. 33 

Table 5. — Numbers oj bacteria xvith distribution in different groups in cream. 



No. 


Bacteria per 
cubic centi- 
meter. 


High acid. 


Low acid. 


Alkali. 


Pepton- 
izers. 


1 
No change. 


1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
11 
12 
13 
14 
15 
16 
17 
18 
19 
20 
21 
22 
23 
24 
25 
26 


116,000,000 
61,000,000 
23,500,000 
40,000,000 
99,000,000 
34,000,000 
11,800,000 
33,500,000 
51,000,000 
40,000,000 
8,000,000 
21,800,000 
37,000,000 

101,000,000 

147,000,000 
54,500,000 
69,500,000 
30,000,000 
89,000,000 
98,000,000 

110,000,000 
45,000,000 
52,000,000 
87,500,000 
39,500,000 
5,700,000 


Per cent. 
25.8 
41.6 
21.2 
48.8 
14.1 
36.1 
16.3 
49.0 
27.1 
9.3 
27.9 
20.4 
27.8 
68.7 
60.2 
36.0 
29.5 
51.7 
42.9 
15.3 
15.0 
12.6 
30.0 
6.8 
7.5 
33.3 


Per cent. 
11.3 
16.6 
7.7 
2.4 
9.9 
8.4 
12.1 
23.0 
14.6 
2.3 
8.9 
3.3 


Per cent. 
1.0 
6.7 
1.0 


Per cent. 

1.0 
10.0 

7.3 
12.2 
25.3 


Per cent. 
60.8 
25.0 
62.1 
36.6 
50.5 
55.6 
55.9 
25.7 
54.2 
80.0 
55.7 
70.3 
61.1 
18.7 
24.7 
46.0 
47.5 
24.1 
46.2 
84.7 
78.0 
68.5 
46.0 
87.5 
80.0 
64.9 


1.1 


1.7 


12.9 
3.0 
4.2 
7.2 
1.3 
6.4 
8.3 
4.4 
1.8 
6.0 
3.2 
3.4 
1.1 






6.3 


2.8 
4.4 
5.3 
6.0 

8.2 
3.4 
2.2 


3.6 
8.0 
6.0 
11.5 
17.2 
7. 7 


7.0 
18.9 
12.0 
5.7 
7.5 
1.8 












12.0 






5.0 









It will be noticed that the number of bacteria in the cream was 
high and that there Avas great variation in the proportion of the groups 
from day to day. 

REINOCULATION OF CREAM. 

Typical cultures were saved from each group and used to inoculate 
one-half of a lot of pasteurized cream. Sufficient amounts of milk 
cultures were added to the pasteurized cream to bring the bacterial 
count at the end of about 24 hours to numbers and proportions 
approximately those of the cream before pasteurization. This was 
of course very difficult to control, but the results given in Table 2 
show that this was attained within reasonable limits. Two experi- 
ments of this kind were made, each consisting of three lots of butter. 
One-third of a lot of sweet cream was cooled and churned; two- 
thirds was pasteurized by holdmg at 145° F. for 20 minutes; one- 
half of the pasteurized cream was cooled and churned, and the 
remaining portion inoculated with the milk cultures and held about 
24 hours to allow bacteria to develop. There was no appreciable 
increase in acidity in this period. The cream was then cooled and 
churned as before. It is probable that the lipase was destroyed and 
the galactase weakened. Tests showed that catalase was present in 
the raw cream, absent in the pasteurized cream, and present again 
in the inoculated cream, while all three lots gave a test for peroxidase. 
66711°— Bull. 162—13 5 



34 



CHANGE IN FLAVOE OF STORAGE BUTTER. 



The score of the butter given in Table 7 shows that while there 
was a marked difference in the rate of deterioration in the raw-cream 
butter and the pasteurized-cream butter, the reinoculation of the 
cream with bacteria had little or no effect on the keepmg quality of 
the butter. In one case the inoculated-cream butter changed more 
than the uninoculated, while in the other lot the reverse was true. 

Table 6. — Bacteria in cream used /or making butter. 
No. 1. 



Treatment of cream. 



Bacteria per 
cubic centi- 
meter. 



High 
acid. 



Low 
acid. 



Alkali. 



Pepton- 
izers. 



No 
change. 



Raw 

Pasteurized 

Pasteurized reinoculated . 



48,000,000 

53,000 

20,000,000 



Per cent. 
13.1 



Per cent. 
10.9 



Per cent. 
0.0 



Per cent. 
17.4 



Per cent. 
58.6 



2.5 



72.2 



Kaw 


47,000,000 

23,400 

217,000,000 


46.0 


6.3 


0.0 


6.3 


41.4 








26.8 


.5 


1.0 


1.0 


70.7 







Table 7. — Scores oj butter — raw, pasteurized, and reinoculated cream. 





First score. 


Second score. 


Treatment of cream. 


Age, 
days. 


Total 
score. 


Comments. 


Age, 
days. 


Total 
score. 


Comments. 


Raw 


6 

6 
6 

4 


86 

92 
91 

86 
92 

91 


Very unclean, some- 
what rancid. 

Slightly woody 

Woody 


38 

38 
38 

36 
36 

36 


82 

87 
91 

82 
90 

89 


Rank, woody, rancid. 




Very woody. 




Somewhat woody. 


lated. 
Raw 


Unclean, rancid 

Slightly woody 


Rancid, woody. 


Pasteurized 


4 


Slightly unclean, 




4 


woody. 
Woody. 


ated. 







It is perhaps significant that raw, sweet-cream butter always 
becomes rancid, a flavor usually associated with the liberation of 
fatty acid. It is not improbable that the lipase of the milk is able to 
act under these conditions, but that the ripening of raw cream, which 
usually prevents the development of this flavor, mhibits the action 
of this enzym. 

THE POSSIBLE OXIDATION OF BUTTER BY INCLOSED AIR. 

The possible oxidation of butter by finely divided ah globules 
mclosed m it, as previously pointed out, made it desirable to find 
out how much air was present in butter exclusive of large pockets, 
and second, whether the air present in butter underwent a change 
during sitorage through the transfer of oxygen from the air to some 



THE POSSIBLE OXIDATION OF BUTTER BY INCLOSED AIR. 



35 



oxidizable substance in the butter. The work was done in the field 
laboratory of the Dairy Division, Troy, Pa. 

Method of gas analysis. — The following diagram shows the appa- 
ratus used for obtaming the gas from a can of butter. The deter- 
mination of the amount of ah (or gas) in a can of butter was made 
as follows : The can of butter was removed from the refrigerator late 
in the afternoon and allowed to remam at room temperature over 
night. The next morning the can was placed in water at 45° C. for 
about an hour unt'd the contents of the can were melted. The flasks, 
previously connected, and the Toepler pump,^ were then evacuated 
by the Geryk pump through the tube d. Tube /could be evacuated 
by opening the stopcock toward the Toepler pump. The Geryk 
pump was worked until the McLeod gauge on the Toepler pump 
read about 0.3 millimeter. The flask i and tube / were then evac- 



"^ To Geryk pump 




Fig. 1. — Apparatus used for obtaining gas from a can of butter. 

uated. The can punch was then brought under tube g and the 
can of butter placed mside of n. The brass plate g, carrying the 
upper needle, was placed over the can. The plate is held over the 
can by two brass guide rods, threaded at their upper ends and 
screwed mto r, the brass base, g was then screwed down a short 
distance, so that the upper end of the upper needle I could be brought 
under the lower end of tube g. I and g were connected by the 
usual rubber tube and rubber nipple mercury seal. Water at 60° C. 
was poured into the contamer until the can was completely sub- 
merged. The water was then colored by adding a few cubic centi- 
meters of alcoholic solution of methylene blue. Previous to puttmg 
the can of butter into n, the funnel p was filled with a three-fourths 
saturated sodium chlorid solution at 50° C. and air bubbles removed 
from 0, partly by squeezing o and forcing the air bubbles up to p, 

1 We used an improved form of Toepler pump designed by Dr. Clark, of this laboratory. 



36 CHANGE IN FLAVOR OF STORAGE BUTTER. 

and then by opening the screw chxmp at t and allowuig the solution 
to flow through V hito n. The two needles were forced into the can 
by screwing down q. In order not to open the mercury seal at the 
upper end of I, the entire punch was raised as fast as I was lowered, 
by means of several thin wooden strips and a wedge on both sides. 
When it was apparent that the rubber stoppers Tc were pressing very 
tightly against the can, and very soon the needles would punch the 
can, I and g were evacuated by openmg the stopcock toward i. 
The enthe system was now empty, from I to g,f, c, b, and the Toepler 
pump. The Geryk pump was then cut off altogether from the rest 
of the system, q was still further forced down until the lower and 
then the upper needles pierced the can. When the upper cover was 
pierced, butter fat would fill tube g to stopcock Ji'. This was then 
opened and butter fat was slowly passed through / into c, where it 
remained. The inclosed gases passed on through h to the Toepler 
pump. The screw clamp t was removed soon after the butter began 
to flow out of the can, permittmg salt solution to enter the can as 
fast as butter left it. Durmg the passage of butter fat, and later of 
butter curd solution, the entire can was covered with water. In only 
one expermient did any colored solution find its way into the cube g. 
Stopcock h' shut off the connection between g and /when salt solution 
began to come through g. This usually happened when most of the 
butter fat and part of the curd solution had passed into c. Knowmg 
the weight of the can of -butter, the empty can, the weight of c 
empty and when fiUed wdth butter from the can, the amount of 
butter passed mto c could be ascertained approximately. In general 
between seven and eight tenths of the contents of the can were passed 
into c during the experiment. The gas m the Toepler pump and 
flasks was then pumped out in the usual way and measured. Part of 
it, 50 to 60 cubic centimeters, was used for the determination of car- 
bon dioxid and of oxygen. The usual apparatus was used. The 
determinations were made according to the methods described in 
Hempel's Methods of Gas Analysis, 1906, pages 149 and 201. 

One blank on air was made before the results were obtamed, one 
after, and two between results, so that the reagents were certainly in 
good condition. 

In the third determination on 2A a few drops of colored water were 
seen to pass up the tube g. No more butter (butter fat) was allowed 
to pass mto c and the estimation of the quantity and composition of 
the gas was made as usual. It is probable that the apparently high 
carbon dioxid was due to gi'eater volatilization of acid -react mg 
material. In this experiment flask c contained 525 grams of butter 
instead of the usual 700-800 grams. 

Results. — The butter used had been churned from pasteurized 
ripened cream, packed carefully in 2-pound thi cans, and sealed by a 



THE POSSIBLE OXIDATION OF BUTTER BY INCLOSED AIE. 



37 



sealmg machine. Determinations were made before and after stor- 
age. Part of the butter was worked normally in the churn (samples 
marked ''A"), part was overworked (samples marked "B"). Sam- 
ples lA and IB were obtamed from the same lot of butter and samples 
2 A and 2B were from another lot. 

Table S. — Quantity and composition of gas in pastexirized riperied-cream butter. 

BEFORE STORAGE. 





Volume of gas 
obtamed from 
1 can (capacity 
approximately 
1 liter) of but- 
ter reduced to 
0°C. and 760 
mm. of mer- 
cury. 


Composition of gas. 


Lot No. 19.4. 


Carbon 
dioxid. 


Oxygen. 


Sample No.— 

1 A (normal) 


. c. 

99.6 
101.4 
107.7 

98.8 
141.5 
104.6 
101.4 


Per cent. 


Per cent. 


IB (overworked ) 






lA 


31.3 
29.7 
36.8 
37.8 
37.8 
.8 


19 1 


IB 


20 3 


2A 


19 1 


2B 


19 5 


2B 


18 6 




21 87 









AFTER STORAGE. 



Sample No.— 

lA 


94.1 

76.9 

139.0 

112.0 

71.0 

117. .5 


34.2 
32.5 
31.5 
39.3 
52.8 
33.6 
.38 
.39 
.39 
.66 


13 3 


IB 


11 


2A... . 


13 1 


2A 


12 1 


2A... 


10 


2B 


13 3 


Blanks on air 


22.1 






21.5 
20.77 

21.82 



The results (see table 8) before storage were obtained in November 
and December, 1911. All of the results for carbon dioxid were 
obtamed on the same pipetteful of potassium hydroxid solution and 
all of those for oxygen on the same pipetteful of sodium pyrogallate. 
The blank was made after all the other results had been obtained. 
Two separate determinations on two cans of 2B showed that the same 
result could be obtamed on two cans of the same lot of butter. 

On the assumption that the gas in butter is a mixture of carbon 
dioxid and aii-, the oxygen content found is apparently high. 

If the percentage of carbon dioxid be subtracted from 100, leaving, 
for example, 70 per cent of the gas supposedly an, the percentage of 
oxygen (20) forms too large a part of the total volume of gas from 
which carbon dioxid has been removed. 

The butter was stored at 0° F. until the following March, 1912, 
during which month the results after storage were obtained. 

The results are difficult to interpret, partly because very few of 
them have as yet been obtained and partly because certain check 



38 CHANGE IN FLAVOR OF STORAGE BUTTER. 

experiments not yet made will be necessary. There seems to be n( 
great variation in carbon dioxid content, but a decided lowering of 
the oxygen content as if part of this gas had been removed. It is 
possible that further work will show that the oxygen in these experi- 
ments was actually transferred to some oxidizable substance in the 
butter. It would perhaps be better to defer conclusions until further 
work shows that the oxygen, if it was indeed removed, went to some 
butter constituent and was not combined with metal or diffused out 
tln;ough the seals. It seems highly desirable to ascertain definitely 
whether cans sealed by a machine without the use of solder are gas 
tight, me;asurmg gas tightness for a period of several months. 

The cans used were heavily tinned and well lacquered on the inside. 
The rims of the covers were provided with a layer of rubber cement, 
which was forced into the seal by the sealmg machine. Cans sealed 
in this way are air tight when tested under 15 pounds' pressure for a 
short time. 

The figures for the composition of the gases in butter obtained after 
storage show that the gas is apparently a mixture of carbon dioxid 
and air. At present any conclusion regarding the nature of the gas 
is premature. It seems almost certain that the gas m these samples 
was not a mixture of carbon dioxid and air only, but that some vola- 
tile substance was mixed with it. The gas obtained from butter had 
an intensely "buttery" odor. Perhaps a more detailed analysis of 
the gas will make the results more intelligible. Under the cii'cum- 
stances it is not safe to assume that the figures for carbon dioxid 
represent carbon dioxid alone, for any substance having an acid 
character and volatile under the conditions of the experiment would 
probably be included with the carbon dioxid in the potassium 
hydroxid absorption. These results show that butter contams 
about 10 per cent by volume of gases. 

Overworked butter did not contain any more air than that which 
had been normally worked. For obvious reasons these results are 
not comparable with those obtained by overworking small amounts 
of butter with a spatula.^ It may, however, be significant that the 
decrease in oxygen as shown in Table 8 was about 50 per cent gi-eater 
in the overworked sample, IB, than in any other. 

THE EFFECT OF METALS ON BUTTER. 
EARLIER INVESTIGATIONS. 

The influence of metals on the changes in butter has received some 
attention, although most of the experiments along this line have not 
included storage butters. In 1902 Henzold - found butter with 

1 Rogers, L. A. Fishy flavor in butter. United States Department of Agriculture, Bureau of Animal 
Industry, Circular 146. Washington, 1909. 

2 Henzold , Ottomar. Bittere Butter. Mileh-Zeitung, vol. 31, no. 52, pp. 822-823, Leipzig, Dee. 27, 1902. 



THE EFFECT OF METALS OK BUTTER. 39 

a bitter astringent taste, which he concluded was caused by iron m the 
sah. He made butter from pasteurized cream, to which salt con- 
taining 0.05 to 0.1 per cent iron oxid was added. The butter 
made, using this salt, had a decidedly bitter taste. By elimination 
and control of conditions, the iron was found to be the factor influ- 
encing this taste. 

L. Marcas ^ showed the effect of holding milk and cream in rusted 
cans for from 2 to 46 hours and making butter from the cream 
treated in this maimer. He determmed the amount of iron in the 
mUk, skim milk, cream, butter, and buttermilk, holding part in a 
clean can and the other in a rusted can for comparison. He found in 
all cases a bitter, astringent taste and bad odor in butter made from 
milk held in rusted cans, while the butter made from the milk held in 
clean cans was of good quality. He concluded that the mUk commg 
in contact with the iron rust forms iron lactate from the iion oxid and 
that it is the lactate which causes the bitter taste. This solvent 
action he found to be especially active in cieam, owing to its acidity. 
He found that cream with a normal iron content of 0.005 parts per 
1,000 would mcrease to 0.240 by 22 houre' contact with a rusted can 
and to 0.270 by 46 hours' contact. Butter made from the cream 
containmg 0.240 parts per 1,000 contained 0.080 parts of iron per 
1,000, while butter made from cream containmg 0.270 parts per 1,000, 
contained 0.134 parts of iron per 1,000. 

Hoft - m 1909 added iron salts (ferrous ammonium sulphate and 
iron lactate) to cream, allowed the cream to stand up to 22 hours, 
made butter, scored it for physical changes, and made qualitative 
tests for iron iti the butter. He added iron in quantities rangmg from 
2 parts per 1,000,000 to 33 parts per 1,000,000. He published results 
of only eight tests, which showed m most cases an oily metallic taste 
in the butter and in which the presence of ii*on in the curd solution 
was determined with potassium sulphocyanid. He, however, cau- 
tioned against definite conclusions, as he had not decided that the 
change was due to iron unconditionally. He found that small 
amounts of iron acting for a long time caused more effect than large 
quantities of iron for a short time. 

In 1911 the Molkerei-Zeitung ^ published some work on the effect 
on butter of washing it with water containing 9 to 15 milligi-ams iron 
per liter. This caused the butter to have a metallic, oily, tallowy 
taste. The butter when washed with water from which the iron had 
been removed by oxidation and filtration did not show the faults 

1 Marcas, L., and Huyge, C. Influence de la rouille sur la quality du beurre. L'lndustrle Laitifere, 
vol. 30, no. Ifi, pp. 187-188. Paris, Apr. 16, 1905. 

- Hoft, Dr. Kann man aiLs dem chemischen Nachweis von Eisen in der Butter auf eine Qualitatsver- 
minderung der Butter durch das Eisen schliessen? Milchwirtschaftliches Centralblatt, vol. 5, no. 6, pp 
250-252. Leipzig, June, 1909. 

3 Entelsenungsanlage fiir Molkereiwasser. Molkerei-Zeitung, vol. 25, no. 58, pp. 1095-1090. Hildesheim, 
July 28, 1911. 



40 CHANGE IN FLAVOR OF STORAGE BUTTER. 

mentioned. It was also found that holding cream in rusted vessels 
caused this oily, metallic taste. 

Kooper/ in 1911 did some work to show that washing butter 
with water containing pure metallic iron would not, in quantities up 
to 36 milligrams per liter, cause any noticeable changes in the quality 
of the butter, but that the changes that took place were caused by 
othei substances in the water together with the iron. He is of the 
opinion that water containing a high percentage of iron is very likely 
to contain H2S or nitrous acid, which would be more likely to cause 
the changes and defects in the butter than the iron itself. Kooper 
used a saccharated iron carbonate in his work. He says, however, 
that a change of the iron to lactate is caused by contact of high acid 
cream with iron, and this will produce the oUy and metallic flavors. 
According to Kooper the iron taken up depends on the length of time 
of contact and the acidity of the cream. By adding rusted naUs or 
pulverized iron rust to the cream before ripening and allowmg the 
cream to ripen in contact with the iron rust he found the cream to be 
changed to a grayish color and showing defects in odor and taste. 
He also showed that the washing tended to take some of the iron from 
the butter. 

A summary of the preceding work, then, shows that some investi- 
gators think the iron in wash water (9 to 15 milligrams per liter) is 
responsible for the changes in butter, while others do not think the 
iron alone (up to 36 milligrams per liter) will cause the changes. All 
seem to agree that the contact of acid cream will take up iron from 
rusted containers, change it to a lactate, and produce butter of poor 
flavor. 

In order to see whether butter containing iron would change more 
in storage than clean butter, it became necessary to make butter 
which contained no mipurity or foreign matter other than iron and 
also to make a control butter containing no foreign material and 
free from iron. 

Anyone familiar with creamery methods will readily see that to 
make butter free from contact with iron would require careful super- 
vision of the cream from the time of milking to the making of the 
butter. In order to do this, the cream was all selected from farmers 
who were careful in the handling of the cream and whose cans were 
free from rust, as this is the first opportunity for contact with iron. 
The cream was, in most cases, pasteurized in a Jensen flash pasteur- 
izer at 75° C. This was carefully cleaned. The cream was cooled, 
weighed, and then ripened in enamel-lined tanks free from iron. 
These tanks were made especially for this work to eliminate any 
chance for contact with iron during ripening, at which time, owing 

1 Kooper, W. D. 1st der Eisengehalt des Wassers von Einfluss auf die Qualitat der Butter? Milch- 
Zeitung, vol. 40, no. 29, pp. 285-287. Leipzig, July 22, 1911. 



THE EFFECT OF METALS ON BUTTER. 41 

to the acidity of the cream, the cream would be most likely to attack 
and dissolve the iron. A pure culture starter was used, the same 
precautions in regard to metals being taken in the preparation of the 
starter. After pasteurizmg, the cream was divided into two portions, 
each being put into an enamel-lined tank for ripening. The cream 
was cooled by running brine through tinned copper coils so suspended 
in the vats that they could also be used for stirring and mixing. 
The cream in one vat was held under normal conditions and free 
from iron. To the cream in the second vat there were added known 
amounts of iron. Both ferrous sulfate and ferrous lactate were 
used in this work. This iron was added in amounts varying from 
1 to 500 parts of iron per million of cream (or 1 to 500 milligrams of 
iron in a kilo of cream). The two vats of cream were then ripened 
under like conditions and churned at the same time in the Disbrow 
combined churns and workers (B2, of 50 gallons capacity). These 
churns were shipped to Troy, Pa., from Albert Lea, Minn., in the 
spring of 1911 and were exposed for some time. Before using at 
Troy they were taken apart, scraped, sandpapered, and thoroughly 
cleaned, though in spite of this one iron plate at the side showed 
rust which was exposed to the cream. The butters were worked 
the same amount, salted the same, and washed with the same amount 
of water and the same number of revolutions in wash water. This 
was considered an essential point, as Kooper found that butter that 
was washed and worked held less iron than that which was worked 
without washing. The butters were then carefully packed in glass 
jars, with glass tops, to avoid any contact with metals, and placed in 
storage. The butters made at Albert Lea, ^linn., were scored about 
a week after making and again after three months' storage at 10° F. 
This butter was scored by J. B. Neumann, J. C. Joslin, P. H. Keiffer, 
and the Fox River Butter Co., none of whom were familiar with the 
history of the butter. As these butters were shipped to several cities, 
it was impossible to get scores on the day the butters were made, 
the time usually approximating one week. 

The butter made at Troy, Pa., was scored by Mr. Fryhofer and 
Mr. Smarzo, of New York City, after from two to four days, again 
after about a month, and again after about three months' storage at 
from 6° to 10° F. By comparison of the scores of the control butters 
and those to which iron had been added at the different intervals the 
effect of the iron on the quality of the butter was determined by the 
score for flavor, aroma, and body. 

In order to determine the relation between the amount of iron 
present in the butter and the deterioration of the butter, it became 
necessary to know the amount of iron present, as well as the changes 
shown by the scores. 



42 CHANGE IN FLAVOR OF STORAGE BUTTER. 

The very small amount of iron present in normal butter (averaging 
1.33 milligrams per kilo for 10 samples from Troy Creamery Co.) 
necessitated the use of a very delicate method. The colorimetric 
method for iron with potassium sulphocyanid is, according to Neu- 
mann,^ so delicate as to show 1 part of iron in 1,600,000 parts of water. 
This method was employed for the determination of the iron. 

METHOD OF ANALYSIS. 

The method of analysis used is as follows: About 500 grams of 
butter are melted and the clear supernatant fat separated from the 
curd solution. At first the fat was disregarded and only the curd 
solution used for analysis. Later the fat, too, was analyzed and 
found to take up approximately 10 per cent of the total iron m the 
butter, so that in all later work the fat and curd were both analyzed 
for iron content. The curd solution is evaporated almost to dryness, 
the remaining fat burned off, and then ashed at a low heat. The fat, 
by heating to its ignition pomt, will ignite and burn; the residue con- 
taining some small pieces of curd which remained in the fat is ashed. 
This ash, when total iron is wanted, is added to the ashed curd and the 
whole extracted with hot dilute hydrochloric acid and filtered into 
a graduated 300 cubic centimeter flask. It may be necessary to 
extract several times, and perhaps even burn the filter and residue 
and again extract in order to be sure to get out all the iron. In 
order to avoid any contamination with iron, this process of ashing is 
carried out in platinum dishes and a platinum spatula used in stirring 
or scraping the ash from the dish. These separate extracts and 
washings are all filtered mto the same volumetric flask and diluted 
to 300 cubic centimeters with iron-free distilled water. This solution 
is then oxidized to have all iron in the ferric condition and an aliquot 
portion (from 0.5 to 10 cubic centimeters, depending on the iron 
present m the solution) transferred to a 50 cubic centimeter Nessler 
tube. Then 5 cubic centimeters of a 10 per cent solution of potassium 
sulphocyanid are added and the whole diluted to 50 cubic centimeters 
with distilled water. These samples are then compared with a set of 
standards made by using known amounts of a solution containing 
0.0001 gram iron per cubic centimeter and made up to 50 cubic 
centimeters in the same way as the unknown. These standards fade 
on standing, so they should be compared shortly after the standards 
and unknowns are made. The results are calculated to parts per 
million (milligrams per kilo). 

By such an analysis the actual amount of iron in the butter samples 
is determined. In most cases the iron was added to the cream, and 

1 Neumann, B. Die Grenzen der Empflndlichkeit verschiedener Reactionen auf Metalle. Chemiker- 
Zeitung, vol. 20, no. 79, pp. 763-764. Cothen, Sept. 30, 1896. 



THE EFFECT OF METALS ON BUTTER. 43 

although the conditions were kept as nearly alike as possible, still 
a difference in the consistency of the butter when washed, or the 
thoroughness and length of the draining and washing would make a 
difference in the amount of iron retained in the butter. 

Notwithstandmg the fact that care was taken to get the best possi- 
ble cream, the iron content can not be expected to be so low as in 
cases where the milk is drawn mto glass vessels and that portion 
used for analysis. The normal creams for about 34 samples had 
an average iron content of 1 .53 milligrams per kilo, with a maximum 
of 3.8 and a minimum of 0.45. Only 6 samples showed over 2 
milligrams per kilo. These samples were taken, some at Albert I^ea, 
Minn.,, some at Troy, Pa., and some at Washington, D. C. 

Relation of iron in butter to iron in the cream. — ^Kooper,^ in his 
investigations found that by washing and working butter it would 
lose some of its iron content. In this work, although care was taken 
to add the same relative amount of wash water and to work and 
wash the butter with the same number of revolutions, the iron found 
in the butter did not show any definite relation to the amount of iron 
added to the cream. In the first series of experiments analyses were 
made of the cream, butter, buttermilk, and wash water in an effort 
to determine whether there was any definite relation between the 
amount of metal added to the cream and that found in the butter, 
buttermilk, and wash water, but, as will be seen by the following table, 
there was no uniformity in the results. The analyses were made on 
samples of about 500 grams and calculated to milligrams per kilo. 
The total amount of butter, buttermilk, and wash water were not 
weighed accurately, and so the total weights of iron in the table are 
only approximated. In the following results the total approximated 
milligrams of iron in butter, buttermilk, and wash water have been 
shown, as well as the percentage return of iron in each of these. A 
percentage comparison, however, unless based on the same amount 
of cream (of same fat content), butter, buttermilk, and wash water, 
and to the cream of which the same amount of iron has been added 
in each case, really does not enable us to draw any definite conclu- 
sions. If the amount of iron added were small, a small variation in 
the amount returned would make a very much larger percentage 
difference than if the same variation were shown on a larger addition 
of iron. This would be especially marked on controls, the cream of 
which would have from 1 to 2 milligrams of iron per kilo, while the 
butter in controls, however carefully made, would show as much or in 
most cases more than the cream, to say nothing of the amount of iron 
found in the buttermilk and wash water. The possible error of 

•Kooper.W. D. Loc.cit. 



44 



CHANGE IN FLAVOR OF STORAGE BUTTER. 



sampling when working on these small amounts also tends to mini- 
mize the value of the percentage relations. 

Following are some of the results as found, the milligrams per kilo 
being accurate but the total weights of iron only approximated : 



Table 9. 



-Relation between iron added to cream and the ironjound in butter, buttermilk, 
and wash water. 



Butter 
No. 


Cream. 


Butter. 


Buttermilk. 


Wash water. 




Total 


Iron 


Total 


Total 


Per- 
centage 


Total 


Per- 
centage 


Iron 


Total 


Per- 
centage 




added. 


iron 
added. 


found. 


iron. 


iron 
found. 


of added 
iron. 


iron. 


of 
added 
iron. 


found. 


iron. 


of added 
iron. 




3fgs. 




Mgs. 




Per 


Mgs. 




Per 


Mgs. 








per kilo. 


Mgs. 


perVdlo. 


Mgs. 


cent. 


per kilo. 


Mgs. 


cent.. 


per kilo. 


Mgs. 


Per cent. 


8 


1,000 


2, 736. 


180. 90 


172 


6.3 


1 , 189. 00 


2,310.0 


84.40 


17.70 


35 


1.30 


13 


500 


1,310.0 


84.00 


77 


5.9 


510. 20 


962.0 


73.40 


36.40 


73 


5.60 


19 


200 


9,613.0 


29.40 


402 


4.2 


69.60 


2,554.0 


26.57 


92.60 


4,222 


42.92 


27 


100 


4,808.0 


11.40 


140 


2.9 


61.20 


2,252.0 


46.85 


2.90 


137 


2.90 


38 


50 


2,296.0 


4.29 


66 


2.8 


24.10 


914.0 


39.80 


15.60 


711 


30.97 


81 


50 


2,269.0 


15.60 


212 


9.3 


39.40 


1,253.0 


55.22 


1.20 


27 


1.20 


51 


20 


862.0 


7.78 


85 


9.9 


8.89 


304.0 


35.27 


.40 


10 


1.20 


155 


20 


862.0 


8.60 


112 


13.0 


18.90 


605.0 


70.20 


7.20 


164 


23.95 


43 


(.^) 


40.0 


3.03 


33 


82.5 


2.17 


58.7 


146.30 


1.44 


33 


82.50 


85 


(') 


103.2 


2.22 


39 


37.8 


1.73 


75.0 


72.70 


.97 


22 


21.30 



> Iron in contact twenty minutes. = Control. Found 1 milligram per kilo. 

3 Control. Found 1.98 milligrams per kilo. 

Although from i..iese results no definite relationship can be stated 
between the amount of iron added to the cream and the amount of 
iron found in the butter, buttermilk, and wash water, yet in a general 
way it might be stated that a relatively small part of the iron goes 
into the butter as compared with the buttermilk, which seems to take 
most of the iron, and in which the presence of a flavor due to the iron 
was most noticeable. 

Distribution of iron between fat and curd solution. — -When this work 
was first started it was thought that the amounts of iron taken up by 
the fat of the butter were negligible, and so in the analyses of butter 
the curd solution only was used for analysis. Later analyses were 
made of the fat as well as the curd solution on 22 samples of butter 
made at Troy, Pa., during the summer of 1911, with the following 
results, basing the fat content in butter at 80 per cent, since all the 
fat was not separated from. the curd solution and the fat contained 
small particles of curd. The fat was not filtered, and so in some 
instances contained small particles of curd, which may account for a 
variance in iron content of the fat. 



THE EFFECT OF METALS ON BUTTER. 
Table 10. — Iron content oj butter, curd, and/at. 



45 





Iron 
added to 


Total 


Percent- 


Percent- 






Butter 


iron 


age of 


age of 


Iron in 


Iron in 


No. 


found in 


total iron 


total iron 


fat. 


curd. 




cream. 


butter. 


in fat. 


in curd. 








Mgx. per 


Mgs. per 






Mgs. per 


Mgs. per 




kilo. 


kilo. 


Per cent. 


Per cent. 


kilo. 


kilo. 


120 





6.64 


7.90 


92.10 


0.66 


30.59 


122 


20 


25.60 


22.70 


87.30 


7.27 


98.80 


125 





6.92 


9.40 


90.60 


.82 


31.32 


127 


10 


10.99 


3.25 


96.75 


.45 


53.18 


132 





9.11 


4.20 


95.80 


.48 


43.64 


134 


5 


7.20 


10.50 


89.50 


.95 


32.25 


140 





8.24 


8.40 


91.60 


.87 


41.38 


142 


2 


7.40 


12.00 


88.00 


1.11 


32.55 


148 





7.56 


12.00 


88.00 


1.13 


33.27 


150 


1 


8.79 


10.70 


89.30 


1.18 


39.28 


157 





5.45 


15.50 


84.50 


1.06 


23.01 


159 





5.09 


32.40 


67.60 


2.06 


17.21 


164 





4.62 


27.50 


72.50 


1.59 


16.75 


166 





5.71 


17.90 


82.10 


1.28 


23.43 


171 





4.04 


19.60 


80.40 


.99 


16.22 


173 





7.76 


13.10 


86.90 


1.27 


33.70 


193 





4.78 


9.39 


90.61 


.56 


21.73 


194 


(') 


6.33 


7.44 


92.56 


.59 


29.29 


201 





6.77 


8.03 


91.97 


.68 


31.11 


203 


n 


6.93 


10.00 


90.00 


.89 


31.21 


205 





5.18 


8.91 


91.19 


.58 


23.61 


207 


(2) 


9.11 


7.69 


92.31 


.88 


42.07 



1 Rusted can. 



2 Iron strip. 



In this table, as in any table of this kind, the percentage relation 
is relatively unimportant. The question of the actual amount of 
iron found in the fat is of much greater importance. An average 
of the control butters, of which there are 14 in the above table, will 
show a content of 1 milligram of iron per kilo of butter fat, a very 
small amount that could be disregarded m most cases. In normal 
butters made under proper conditions where the total iron content 
is about 1.3 milligrams per kilo, even a difference of 0.5 milligram 
of iron in a pound of butter would be noticeable on a comparative 
basis between the fat and curd content, but when total iron is being 
considered so small an amount as 0.5 milligram of iron could easUy 
be discarded in the calculation. 

THE INFLUENCE OF IRON ON FLAVOR. 

The ideal condition for the solution of this problem would of 
course be to have butter made absolutely free from iron while known 
amounts of iron, or any other metal as the case might be, could be 
added to a portion of the cream used in making this metal-free 
butter. This could be done only by drawing the milk samples 
directly mto glass vessels, skimming by hand, and ripening and 
churning m small quantities in glass. This method of procedure 
seemed impracticable, since enough butter could not be made to 
satisfy all the requirements of the experunent. The first few sam- 
ples were, however, churned m a tall glass jar by shakmg by hand, 
but proved unsatisfactory in that only a small amount could be 



46 



CHANGE IN FLAVOR OF STORAGE BUTTER. 



made and the butter was not very good. The best thing to be 
done under the circumstances was to control conditions as carefully 
as possible and avoid any undue contact with iron during the whole 
process of butter making. The method of butter making has been 
described. The butters were scored for the first time in most cases 
very shortly after making, being kept in cold storage until scored. 
It will be noticed that the butters made at Albert Lea, ]\Iinn., were 
scored by the Fox River Butter Co. and those made at Troy, Pa., 
or Washington, D. C, were scored by Mr. Fryhofer and Mr. Smarzo, 
of New York City. For this reason these scores are not comparable, 
as in our experience there is considerable variance between the 
scores and methods of scoring even among professional butter 
scorers. Following is a table showing the butter scores. In every 
case the butter to which metal has been added is followed by a con- 
trol butter, made at the same time under the same conditions. 

Table 11. — Influence oj iron on flavor oj butter. 



Data on ripened, pasteurized cream. 



Butter 
No. 



Iron 

content 

normal 

in cream. 



Iron 

added as 

ferrous 

sulphate. 



Dura- 
tion of 
con- 
tact. 



Acidity 
at time 
of churn- 
ing. 



Iron 
found 



butter. 



Age of 




butter 
when 


Score. 


scored. 




Days. 




7 


85.0 


187 


85.0 


7 


92.5 


187 


85.0 


11 


92.0 


184 


85.0 


11 


93.5 


184 


85.0 


14 


86.0 


179 


85.0 


14 


87.0 


179 


85.0 


9 


87.0 


175 


85.0 


9 


92.0 


175 


86.0 


16 


93.5 


160 


88.0 


16 


94.0 


160 


88.0 


3 


86.0 


44 


85.0 


116 


82.0 


3 


93.0 


44 


84.5 


116 


80.0 


5 


84.0 


42 


85.0 


114 


84.0 


5 


88.0 


42 


85.0 


114 


84.0 


3 


91.5 


39 


86.5 


111 


83.0 


3 


94.0 


39 


85.5 


111 


82.0 



119 

'23 

27 

31 

39 

»35 

147 

143 

181 

177 

122 

120 

127 

125 

134 
132 



Mgs. per 
ikilo. 



10.90 

3.84 

3.84 

.40 

.40 

.99 

1.00 

2.76 

2.76 

2.93 

2.90 

2.11 

2.48 

1.64 
1.64 



Mgs. per 

kilo. 

200 


100 


50 


25 


50 


20 



10 



6 




214 



Per cent 
of lactic 



Hours. 
22 


acid. 




0.53 


23 


.77 




.65 


23 


.73 




.73 


23 


.53 





.51 


22 


.60 




.62 


21 


.57 




.58 


23 


.49 




.48 



.59 
.55 



Mgs. per 
kilo. 
29.4 

8.3 

11.4 
2.59 
4.29 
1.70 

26.5 
3.03 

15.6 
2.49 

25.6 

6.64 
10.99 
6.92 

7.20 
9.11 



' Butter made at Albert Lea, Minn. 
Troy, Pa. 



Very fishy. 

Do. 
Coarse, unclean. 
Very fishy. 
Oily. 
Very fishy. 

Do. 
Fishy. 
Very fishv. 
Fishy. 
Very fishy. 
Slightly fishy. 
Very fishy. 
Coarse. 
Very fishy. 
Clean. 
Oily, unclean. 

Fishy. 

Very oily; very fishv. 
Do. 

Rank, oily, and fishy. 

Clean. 

Very fishy. 

High acid, clean, rank fishy. 

Oily, metallic, unclean, rancid. 

OUy, metalUc, very salvy. 

Very oily, metallic. 

OUy, slightly metallic, un- 
clean. 

Oily, metallic. 

Very oily, metallic. 

Metallic, unclean. 

Metallic, stale. 

Very fishy. 

Very clean. 

Fishy. 

Very high acid, clean, very 
fishy. 

Scored by Fox River Butter Co. AU other butters made at 



THE EFFECT OF METALS ON" BUTTER. 4? 

Table 11. — Influence oj iron on flavor oj butter — Continued. 



Butter 
No. 



142 
140 

150 

148 

193 
192 
223 

222 

225 

224 

227 

226 

229 

228 

231 

230 



Data on ripened, pasteurized cream. 



Iron 

content 

normal 

in cream. 



Mgs. per 
kilo. 
1.17 



1.17 
.45 
.74 



2.99 
2.73 



Iron 

added as 

ferrous 

sulphate. 



Mgs. per 
kilo. 
2 





1 



20 



2 10 



25 



2 2.5 



22 



21 





Dura- 
tion of 
con- 
tac. 



Hours. 
17 



Acidity 
at time 
of churn- 
ing. 



Per cent 
of lactic 
acid. 
.59 



.60 
.60 
.60 



20i 



20i 



.668 

. .552 

.66 

.55 

.60 

.54 

.44 

.42 

.46 

.52 



Iron 
found 



butter. 



Mgs. per 
kilo. 
7.40 



8.24 

8.79 

7.56 

5.80 
3.41 
7.37 

3.39 

5.4 

2.15 

3.59 

2.85 

1.61 

1.89 

1.(53 

2.16 



Age of 
butter 
when 
scored. 



Days. 

2 
47 
105 

2 
47 
105 

6 
45 
103 

6 
45 
103 
11 
128 
11 
128 

4 
29 
61 

4 
29 
61 

4 
27 
59 

4 
27 
59 

4 
25 
57 

4 
25 
57 

7 
22 
54 

7 
22 
54 

5 

20 
52 

5 

20 
52 



Score. 



91.0 
87.0 
88.0 
94.5 
88.0 
83.0 
86.0 
86.0 
85.0 
89.0 
87.0 
86.0 
86.0 
86.0 
91.5 
91.0 
84.0 
84.0 
84.0 
86.0 
86.0 
86.0 
88.0 
86.0 
84.0 
90.0 
87.0 
87.0 
87.0 
87.0 
85.0 
93.0 
93.0 
89.0 
88.0 
87.0 
85.0 
92.0 
92.0 
88.0 
94.0 
93.0 
86.0 
92.0 
92.0 
88.0 



Remarks. 



Slightly metaUic. 

Very metallic. 

Oily. 

Slightly metallic aroma, clean. 

Metallic. 

Very fishy, clean, low acid. 

Very oily and fishy. 

Oily and fishy. 

Do. 
Milky 

Slightly fishy. 
Fishy, clean", high acid. 
Oily, very fishy. 
Fishy. 



Unclean, stale, oily. 
Unclean, metallic, gritty. 
Rank, metallic. 
Stale, oily. 
Very oily, metallic. 
Oily, metallic. 

Do. 
Very oily. 

Very metallic and fishy. 
Slightly metallic. 
Unclean, oily. 
Oily, metallic. 
Tainted, very oily. 
Oily. 

Very oily, metallic. 
Good butter. 

Old flavor. 
Very metallic. 
Oily, metallic. 
Very metallic. 
Somewhat oily, coarse. 

Oily, trifle fishy. 
Good, clean, creamy. 

Very fishy. 
Somewhat oily, coarse. 



Little fishy. 



1 Butter made at Albert Lea, Minn. Scored by Fox River Butter Co. All other butters made at 
Troy, Pa. 

2 Iron added as lactate. 

In Table 11 it will be noticed that in every instance on the first 
scoring the butters to which iron had been added scored lower than 
theii" controls. This holds m most cases on the second and third 
scoring, the most noticeable feature being that the butters to which 
iron has been added show the deterioration much faster than the 
control butters. After the butters have deteriorated to a score of 
85 or lower, the butter is so poor that a difference of a point or two 
iij the score really does not indicate a very great difference in the 
quality of the butter. A great many of the butters became fishy, 
and where both were not scored fishy at the same time it wUl be 
noticed that the control butter was the last to become "fishy," though 



48 



CHANGE IN FLAVOE OF STORAGE BUTTEE. 



in one or two instances the control was scored "fishy," while the 
other butter was not marked fishy at all. A very noticeable feature 
about these butters is the production of a very oily flavor. This 
was present in most amples that were marked fishy and seems to be 
a stepping stone to the fishy flavor. 

THE INFLUENCE OF COPPER ON FLAVOR. 

In the work on copper, only the physical changes wiU be con- 
sidered, as considerable difficulty was experienced in making accurate 
determinations of the very small amounts of copper present in the 
butter. 

These butters were made in exactly the same way as the butters 
showing the influence of iron. The cream was all very carefully 
handled to avoid contact with copper, and divided into two portions, 
which were treated in exactly the same manner, excepting that to 
one-half of the cream, copper in the form of a solution of copper 
sulfate or lactate was added in amounts varying from one-half to 
20 milhgrams per kilo of cream. The cream was ripened and churned 
and the butter stored m glass jars without metal parts, or in ash 
tubs, in 1912, and scored at intervals. The changes in the butter 
are shown in the following tables: 







Table 12 


— Influence of copper on butter. 




Data on ripened and pasteur- 
ized cream. 


Butter. 


Butter 
No. 


Copper 
added 

as sul- 
fate. 


Diiration 
of con- 
tact. 


Acidity 
at churn- 
ing. 


Age on 
scoring. 


Score. 


Remarks. 


189 
185 
2 173 

2 171 


Mgs. per 
kilo. 
20 



4 



2 



1 


<5.0 


<2.5 


Hours. 
19 

22 


Per cent 
of lactic 
acid. 
0.51 

.60 

.60 

.60 

.56 

.52 

.51 

.50 

.36 

.385 

.415 


Days. 

14 

158 

14 

158 

5 

38 
96 

5 

38 
96 

2 
40 
98 

2 
40 
98 

4 
42 
100 

4 

42 
100 

3 
18 
50 

3 

18 
50 

2 
15 
47 


90.0 
85.0 
92.5 
86.0 
85.0 
85.0 
80.0 
91.0 
90.0 
86.0 
87.0 
84.0 
80.0 
93.0 
87.0 
87.0 
85.0 
84.0 
80.0 
91.0 
86.0 
82.0 
86.0 
84.0 
84.0 
92.0 
89.0 
89.0 
88.0 
87.0 
84.0 


Very fishy. 

Oily. °' 

Very fishy. 

Oily, fishy, unclean. 

Oily, fishy. 

Rank, fishy, clean, high acid. 

Oily. 


»166 




Slight metallic. 
Fishy, clean, low acid. 
Oily, unclean, rancid. 


2 164 




Very oily, fishy. 

Rank, oily and fishy, unclean, low acid. 

Slight metallic. 


>159 




Oily, metallic. 

Oily. 

Oily, metallic, stale. 


• 157 




Very oilv and fishy. 

Rank, fishy, unclean, low acid. 

Slight oilv. 


8 235 
*234 


19 


Very oily, fishy. 

Very oily, fishy, clean, high acid. 

Very oily and metallic. 

Oilv and fishy. 

Rank, fishy. 

Somewhat oily and coarse. 


3 237 


19 


Oily. 

Old flavor. 
Oily and metallic. 
Unclean, oily. 
Rank, flshv. 



1 Butter made at Albert Lea, Minn., 1910. 
» Butter made at Troy, Pa., 1911. 



3 Butter made at Troy, Pa., 1912. 

■• Copper added in form of copper lactate. 



THE EFFECT OF METALS ON BUTTER. 
Table 12. — Influence of copptr on butter — Continued. 



49 





Data on ripened and pasteur- 
ized cream. 


Butter. 


Butter 
No. 


Copper 
added 
as sul- 
fate. 


Duration 
of con- 
tact. 


Acidity 
at churn- 
ing. 


Age on 
scoring. 


Score. 


Remarks. 


1236 


Mgs. per 
kilo. 


21.5 



2 1.0 



Hours. 


Per cent 
of lactic 
acid. 
.58 

.376 

.355 

.40 
.34 


Days. 

2 
15 
47 

2 
13 
45 

2 
13 
45 

3 
43 

3 
43 


92.0 
91.0 
86.0 
92.0 
92.0 
86.0 
94.0 
94.0 
93.0 
88.0 
87.0 
91.0 
91.0 


Trifle coarse, slightly tainted. 

Slight imclean. 

Tallowy flavor. 

Slight oily and metallic. 

Slight oily. 

Metallic and fishy. 

Good and clean. 


1239 
1238 


m 


« 

1241 
1240 


21 


Clean. 

Very oily and metallic. 

Metallic. 

Trifle metallic. 






Old flavor. 



1 Butter made at Troy, Pa., 1912. 



2 Copper added in form of copper lactate. 



From Table 12 it will be seen that in every instance the scores on 
the control butters were better than the scores on the butter made 
from cream to which copper had been added, even in the small 
amount of 1 milligram of copper per kilo of cream. 

Unfortunately, the ''Remarks" on the butter do not show any 
definite characteristics that can be attributed to the copper. The 
control butters, too, show deterioration in storage, though they seem 
to keep better than the butters made from cream to which the copper 
was added. On the second scoring after 40 days in storage most of 
the butter to which copper had been added showed a fishy flavor 
and after three months a very decided rank, fishy flavor that was 
unmistakable, and was called by scorers decided mackerel flavor. 
These butters were aU made during the summer months. 

An experiment to show the effect of having the cream stand in 
contact with a small surface of copper for a long time was made by 
having a vat of cream ripened in contact with two sheets of bright 
copper each 2 by 6 inches. The sheets were placed on edge in the 
bottom of the vat during the process of ripening. The result of this 
is shown in the following table: 

Table 13. — Effect of copper on flavor oj butter. 



Butter 
No. 


Cream. 


Dilation 
of con- 
tact. 


Age. 


Score. 




Normal 
acidity. 


Churning 
acidity. 


Remarks. 


256 


Per cent. 
0.17 

.17 


Per cent. 
0.355 

.37 


Hours. 


Days. 

1 
31 

1 
31 


93 
91 
91 

88 




257 


21 


Trifle old flavor. 
Trifle oily. 
MetaUic. 



50 



CHANGE IN FLAVOR OF STORAGE BUTTER. 



The difference in flavor is marked in the fresh butter but more 
marked in storage butter. 

Another experiment to show the effect on the flavor of the butter, 
by having the cream come in contact with a large surface of copper 
for a short time, was made by pasteurizing the cream in two parts. 
One part was pasteurized in a No. 3 Peerless pasteurizer, the copper 
lining being completely covered with tin. The second part was 
pasteurized in a No. 5 Peerless pasteurizer, the tin coating of which 
had been worn off by continued usage, leaving the cream exposed 
to a surface of copper during the process of pasteurization. The 
duration of contact with the copper was only a few seconds, though 
the surface was quite large. The temperature of pasteurization was 
about 60° C. 

The results of this experiment are found in the following, table: 

Table 14. — Comparison of the efect oj tin and copper on the flavor oj butter. 







Acidity 
of cream 
before 
pasteur- 
ization. 


Temper- 


Acidity 








Butter 

No. 


Pasteur- 
izer. 


ature of 
pasteur- 


at 
churn- 


Age. 


Score. 


Remarks. 






ization. 


ing. 












Per cent. 


" C. 


Per cent. 


Days. 






245 


Tin 


0.18 


60 


0.55 


2 
39 


91 
92 


Slight oily. 
Good. 


246 


Copper. . 


.18 


60 


.50 


2 
39 


87 
85 


Oily, stale, metallic. 
Very fishy. 
Slight oily. 


252 


Tin 


.19 


60 


.52 


1 


92 












33 


89 


Oily. 


253 


Copper. . 


.19 


60 


.■17 


1 
33 


89 

84 


Do. 

Very fishy. 



These two experiments show very plainly the deteriorating effect 
of poorly tinned pasteurizers, for aside from this all other conditions 
were exactly alike during the complete process of butter manufac- 
ture. Considering the short duration of contact, the change m the 
flavor of the butter even when fresh is very marked. The effect of 
copper even in small amounts seems to cause more marked changes 
in butter flavor than iron, with a marked tendency toward a fishy 
flavor in storage. 

CONTAMINATION OF CREAM WITH IRON FROM CONTAINERS. 

For this experiment a lot of cream was divided into two portions, 
one portion being held in a clean can and the other in a can which 
showed many rust spots. This was the most rusted of all the cans 
that were used for the handling of cream at the Troy Creamery Co., 
of Troy, Pa. However, this showed only small spots of rust on the 
bottom of the can. This raw cream was ripened without the addition 
of a starter at room temperature. The temperature, acidity, and 
iron content are as follows: 



THE EFFECT OF METALS ON BUTTER. 
Table 15. — The absorption oj ironjrom rusty can. 



51 







Clean can 






Rusted can. 


Duration 














of 














contact. 


Temper- 
ature. 


Acidity. 


Iron 
found. 


Temper- 
ature. 


Acidity. 


Iron 
found. 


Hours. 


- F. 


Per cent. 


Mgs. p. kilo. 


° F. 


Per cent. 


Mas. p. kilo. 





66 


0.114 


1.045 


66 


0.114 


1.045 


2 


67 


.101 


1.031 


67 


.120 


1.132 


4 


67 


.146 


1.539 


67 


.158 


1.772 


6 


67 


.177 


1.028 


67 


.176 


1.265 


8 


68 


.202 


1.677 


68 


.212 


1.129 


14 


66 


.412 


.792 


66 


.422 


1.627 


21 


66 


.561 


1.082 


66 


.561 


1.459 


26 


64 


.581 


.951 


56 


.568 


1.187 


32 


52 


.634 


1.082 


57 


.624 


1.257 


48 


44 


.632 


1.185 


57 


.638 


2.381 




Butter m 


ade from a 


30ve cream 


Butter made from 


ibove cream 








4.78. 






6.33. 



Another experiment on the same basis but without the acidity of 
the cream at the various intervals gave the following results, from a 
cream having a normal iron content of 0.9 raiUigram per kilo and an 
acidity of 0.16 per cent lactic acid. • 

Table 16. — Effect oj rusty can on the flavor oj butter. 



Butter. 


Iron in 
butter. 


Cream 
contact 
in cans. 


Iron 

content in 

ripened 

cream. 


Churning 
acidity. 


Butter. 


Remarks. 


No. 


Age. 


Score. 


259 
260 


Mgs. p. kilo. 
1.37 

1.60 


Hours. 
15 

15 


Afgs. p. kilo. 
4.31 

4.35 


Per cent. 
0.51 

.49 


Days. 
19 
29 
19 
29 


92 
89 

88 
86 


Slight oily. 
Trifle metallic, oily. 
Oily, metallic. 
Unclean, oily. 



From Table 15, it is seen, as would be expected, that the acidity 
of cream increases with the length of time it is held, though there is 
no material difference in the rate of increase between the two samples. 
The cream in the rusted can does not show any marked increase in 
iron content over that in the clean can. The difficulty in getting an 
accurate sample of the cream after standing long enough to thicken 
may account for the variation from what might have been expected. 
At the end of 48 hours the cream from the rusted can showed an iron 
content of 1.2 parts per 1,000,000 more than the cream from the 
clean can. The butters made from these creams checked very 
closely, as the butter made from the cream held 48 hours in the rusted 
can showed 1.55 parts per 1,000,000 more than the butter made from 
the cream held 48 hours in a clean can. 

In another instance to find whether cream on standing in contact 
with iron rust would take up iron, a lot of cream was divided into 
four parts, each placed in a clean can, and a strip of rusted iron tape 
one-half inch wide and 24 inches long (giving a surface of 24 square 



52 



CHANGE IN FLAVOR OF STORAGE BUTTER. 



inches) was placed in two of these cans. Two cans, one with and 
one without the iron, were ripened at room temperature for 21 hours, 
and two cans, one with and one without iron, were held in cold 
storage at 32° F. for 21 hours. Butter was made from each of these 
four samples of cream. There was no starter added to this cream. 
The cream from which these four lots were taken had acidity of .223 
and iron content of 1.25 mHUgrams per kilo. 

Table 17. — Data on cream ripened in contact ivith rusty iron. 



No. 


Cream. 


Butter. 


Temper- 
ature. 


Acidity. 


Iron con- 
tent. 


Butter 
No. 


Iron con- 
tent. 


Score. 


1 
12 

3 
14 


"F. 
32 
32 
66 
66 


0.385 
.368 
.655 
.670 


Mgx. per 

kilo. 

1.53 

1.81 

.90 

11.32 


1 
2 
3 
4 


Mqs. per 
kilo. 
6.77 
6.93 
5.18 
9.11 


94 
91 
94 
91 



- In contact with iron tape. 

From this table, although it does not show the acidity and iron 
content at intervals as in Table 15, still it shows here that with 
the development of acidity the iron is taken up by the cream. 
This is very marked in cream and butter No. 4, the iron content 
m both being very high as compared with their respective controls. 
The effect of the iron is also very marked m the butter scores, the 
controls (Nos. 1 and 3) scoring three points higher than the ones to 
which the iron had been added. 

In the first experiment Mr. Larson, superintendent of the Troy 
Creamery Co., said he could taste the iron in the cream after 32 
hours' contact, and in the second experiment the cream in contact 
with iron ripened at room temperature showed a bitter metallic 
taste. 

In every case the butter made from cream which had stood in 
contact with iron rust showed a peculiar taste and was easily picked 
out from a lot of samples. The taste was, however, most noticeable 
iu the buttermUk, to which it gave a decided metallic taste. 

In Table 15 the difference in the amounts of iron found in the 
cream are very small, in only one case being more than 1 milligram 
of iron per kilo of cream. Taking into consideration the possibiUty 
of error in sampling a very thick cream, these small differences do 
not seem to be enough to warrant any definite conclusions as to the 
absorption of iron by the cream. It wUl be noticed in Table 1 1 that 
all the control butters made by us at Troy, Pa., have a very high 
iron content, as compared with control butters made by us at Albert 



THE EFFECT OF METALS ON BUTTER. 53 

Lea or made by the Troy Creamery Co. at Troy. As the wash water 
was tested and the cream did not show a high iron content, the 
churns are the only means whereby iron might be introduced into 
the butter. Although these churns were taken apart and scraped, 
sandpapered, and thoroughly cleaned before being used at Troy, it is 
possible that there were some rust spots on the iron bolt heads and 
plates coming in contact with the cream, and that the iron was 
attacked at these pomts by the high acid cream and the iron taken 
up by the butter. Unfortunately the butter samples were not 
analyzed at the time the butter was made, otherwise this error 
would have been noticed in the chemical results and corrected before 
continuing the work. The high iron content of the butters made at 
Troy, Pa., seemed to show conclusively that there was an error by 
contammation at some s.tep in the process of butter making. The 
only check possible on the butter itself was by analysis of the butter 
made by the Troy Creamery Co. in their large churn. In most cases 
the cream for the experimental butter was selected from the best 
cream brought to the creamery, while the cream used in the creamery 
proper was the regular run of cream as brought in by the farmers 
and from the several skimmmg stations. In this way the butter 
from the creamery proper, if the fault were in the cream, should 
show a higher iron content than the experimental butter. The 
cream used in the experimental creamery was analyzed for iron (in 
most cases) at three stages; first, raw sweet cream; second, immedi- 
ately after pasteurizing (to determine whether there was any iron 
exposed in the pasteurizer or cooler), and, third, after ripening in the 
ripening vats to determme whether there was any iron taken up in 
the vats. The butter was then analyzed, thus giving the last stage in 
the process of butter making. 

The butter made in 1912 at Troy, Pa., was churned in new No. 5 
Bestov box churns, and worked on a table worker, eliminating 
this contamination by iron in the churn. The butter for scoring 
was packed in 10-pound ash butter tubs instead of glass jars in order 
to avoid injuring the body of the butter in packing. 



54 



CHANGE IN FLAVOK OF STOKAGE BUTTEK. 

Table 18. — Iron content oj cream, and controls. 
[Milligrams per kilo.] 



Butter. 


Raw 
cream. 


Pasteur- 
ized 
cream. 


Raw 
ripened 
cream. 


Pasteur- 
ized 
ripened 
cream. 


Curd as 
100 per 
cent of 
butter. 


Ciu-d as 
20 per 
cent of 
butter. 


Fat as 
80 per 
cent of 
butter. 


120 E 
125 E 
1-32 E 
140 E 
148 E 
157 E 
164 E 
171 E 
201 E 
205 E 

135 T 

136 T 
143 T 

151 T 

152 T 

160 T 

161 T 

167 T 

168 T 

195 T 

196 T 

197 T 

198 T 
2 224 

2 226 
•■•228 
2 230 
2 234 
2 236 
2 238 
2 240 
2 246 
2 247 
= 248 
2 252 
2 253 
2 256 
2 257 
2 259 
2 2a3 
2 264 








2.93 
2.57 
1.64 
1.17 
.74 
1.59 
1.63 
1.44 


6.12 
6.26 
8.73 
7. .54 
6.65 
4.60 
3.35 
3.24 
6.22 
4.72 
1.12 
1.16 
1.25 
.96 
1.19 
.89 
1.21 
11..55 
■1.31 
1 1.50 
« 1.79 
' .99 
1 2. 35 
2.15 
2.85 
1.89 
2.16 
2.04 
3.16 
3.50 
3.59 
1.95 
1.36 
1.14 
1.41 
1.41 
3.40 
6.63 
1.37 
2. 69 
1.45 


30.59 

31.32 

43.64 

41.38 

33.27 

23. 01 

16.75 

16.22 

31.11 

23.61 

5.59 

5.81 

6.26 

4.81 

5.97 

4.47 

6.05 

'7.77 

16.52 

17.51 

'8.96 

1 4.96 

1 11. 74 


0.66 

.82 
.48 
.87 
1.13 
1.06 
1.59 
.99 
.68 
.58 


2.11 
1.89 
1.19 
.45 
1.47 
1.46 
1.94 
1.25 
1.75 


2.48 
1.49 
1.12 
.55 






















1.53 
.90 


































































































































:::::::::;:;;;:::;: 




















1 












2.73 














































.94 


2.04 


1 






j 










j 








2.01 
1.14 


1 















1.67 
1.67 










1.10 
.9 






















4.31 
4.35 



























E Butter made in experimental creamerv, Troy, Pa., 1911. 

T Butter made by Troy Creamery Co., 1911. 

' Curd burned in porcelain. 

2 Butter made in experimental creamery, Troy, Pa., 1912. 

Table 18 is arranged to show the u-on content, found in the cream 
and butter made in the experimental creamery and also, for compari- 
son, the iron content of the butters made by the Troy Creamery Co. 
In these analyses the fat was disregarded and the iron found in the 
curd solution used as the total iron in the butter. It will be noticed 
in the column, "Fat as 80 per cent of the butter" that the iron 
content averages 0.89 milligram per kilo. As the usual charge of 
butter was 500 grams, the total u'on in fat would be less than 0.4 
milligram of iron, which is a very small and almost negligible quan- 
tity. 

Ten samples of control butter made in the experimental churns 
gave an average iron content of 5.74 milligrams of iron per kilo of 
butter using the curd solution only and calculating as butter, while 13 
samples of butter made by the Troy Creamery Co. in their large churn 



THEORETICAL CONSIDERATIONS. 55 

during the same period showed an average iron content of 1 .33 milli- 
grams of iron per kilo of butter, using curd solution only and calcu- 
lating as butter. The same cream, with a possible advantage in 
favor of the experimental cream, the same salt, and the wash water 
from the same source, were used in the manufacture of both the experi- 
mental and the Troy Creamery Co.'s butter, so that the only point 
of entry for the increased iron content in the experimental butters 
was in the churns. This might possibly account for the deterioration 
in the control butters made m the experimental creamery in 191 1 , and 
although these results detract from the results in the other experiments, 
still the value of the evidence to show that iron may be taken up in 
the churn is of great importance to the butter maker. Rusty bolt- 
heads, plates, or other castmgs should be carefully guarded against 
by the butter maker. 

THEORETICAL CONSIDERATIONS. • 

Having ascertained definitely, partly from the empirical observa- 
tions of others, and partly from the experimental data* obtained on 
butter containing added iron, that iron itself lowers the keeping 
quality of butter, it was desirable to find out how iron affects butter. 

That the iron acts catalytically in an oxidative reaction at once 
suggests itself. As has been shown, butter made by the usual 
methods contains gases in the proportion of approximately 10 cubic 
centimeters in 100 grams of butter. It seems possible that a few 
parts of iron per million parts of butter, present in a finely divided, 
colloidal condition might be able to transfer slowly some of the in- 
closed oxygen to any one of the oxidizable substances present. It 
is of course well known that in the presence of a peroxid such as 
hydrogen peroxid the iron will rapidly transfer oxygen from the 
peroxid to an oxidizable substance. A transfer of oxygen such as 
this is often referred to as peroxidase action and the substance 
through which the transfer is brought about, the colloidal iron com- 
pound m this case, is called a peroxidase. It is also well known, at 
least the statement is often seen in the literature, that peroxidases 
can transfer oxygen only from peroxids and hence are inactive in the 
absence of peroxids. Kastle,^ in discussing the mode of action of 
peroxidase, states : ' 'According to Bach and Chodat ^ the peroxidases 
exert not the slightest oxidizing action in the absence of the peroxid." 
It is perhaps to be regretted that this statement was not accompanied 
with another to the effect that this conclusion follows from an experi- 

1 Kastle. J. H. The oxidases and other oxygen-catalysts concerned in biological oxidations. United 
States Treasury Department, Public Health and Marine-Hospital Service, Hygienic Laboratory, Bulletin 
59. Washington, 1910. Seep. 117. 

2 Bach, A., and Chodat, R. Ueber Peroxydase. Berichteder Deutschen Chemischen Gesellschaft, vol. 
36, no. 3, pp. 600-605. Berlin, Feb. 21, 1903. 



56 CHANGE IN FLAVOE OF STORAGE BUTTEE. 

ment in which Bach and Chodat ^ made observations on oxygen ab- 
sorption for a period of 24 hours. Bach and Chodat,^ in support of 
their contention that peroxidases are without action in the absence 
of peroxids, quote the work of Linossier ^ which was likewise a series 
of observations on peroxidase action covering only very short periods 
of time.* 

It may be true that peroxidases in the presence of air are inactive 
when their activities are measured for intervals of 24 hours. But in 
the case of cold-storage butter, in which iron and air may interact for 
several months, the possibility that oxidative action may take place 
is not excluded, in spite of the fact that there is at present no reason 
to suppose that peroxids are present in butter. 

The experiments here described on the oxidation of lactose in milk 
through the action of iron salts are in many respects similar to those 
made in recent years by several investigators whose work throws some 
light on the subject. 

Lob and Pulvermacher ^ studied the action of gaseous oxygen and 
of hydrogen peroxid on dextrose and sucrose. They used as an 
oxidative agent a preparation made by extracting pig pancreas with 
alcohol and adding iron to the alcohol filtrate. The precipitate of 
iron-pancreatic material was dried and used in the experiments. In 
24 to 48 hours this substance could oxidize dextrose in aqueous solution 
either in the presence of hydrogen peroxid or by the aid of a stream 
of air or oxygen. Sucrose was also oxidized by hydrogen peroxid 
and iron-pancreas powder, but to a much less extent. They sug- 
gest the necessity of the inversion of the sugar before oxidation can 
take place. 

Battelli and Stern® and Harden and MacLean ^ studied oxidation 
in isolated, hashed animal tissues. These investigators used such 
material as hashed muscle, suspended in water. The suspensions 
were placed in a flask filled with gaseous oxygen. The substance to 
be oxidized was added to the suspension and the flasks were connected 
with an apparatus for measuring the amount of oxygen absorbed. 

According to BatteUi and Stern, succinic acid is very easily oxidized 
in a very few hours under the conditions of their experiment. Harden 
and MacLean repeated some of the experiments of Battelli and Stern 

> Bach, A., and Chodat, R. Loc. cit. See pp. 604-605. 

2 Bach, A., and Chodat, R. Loc. cit. Seep. 605. 

3 Linossier, G. Contribution & 1 'etude des ferments oxydants. Comptes Rendus Hebdomadaires des 
Stances de la Soci6t6 de Biologic, vol. 50, no. 12, pp. 373-.375. Paris, Apr. 1, 1898. 

< The reference to Linossier given by Bach and Chodat is incorrect; the reference by Kastle is correct. 

6 Lob, Walther, and Pulvermacher, Georg. tJber die oxydative ZuckerzerstorungunterderEinwirlning 
von Organpraparaten. Biochemische Zeitschrift, vol. 29, no. 4/5, pp. 316-346. Berlin, Nov. 22, 1910. 

6 Battelli, F., and Stern, L. Die oxydation der Bernsteinsaure durch Tiergewebe. Biochemische 
Zeitschrift, vol. 30, no. 1/2, pp. 172-194. Berlin, Dec. 23, 1910. 

' Harden, Arthur, and MacLean, Hugh. The oxidation of isolated animal tissues. Journal of Physiol- 
ogy, vol. 43, no. 1, pp. 34-45, Sept. 11, 1911. 



THEORETICAL CONSIDERATIONS. 57 

and found that the oxidation of succinic acid was not as vigorous as 
BattelH and Stern had found. 

There are several reasons why the results on peroxidase action 
obtained by one investigator might not be the same as those obtained 
by another on material intended to represent exactly the material pre- 
viously used. The activity of peroxidases is influenced by so many 
conditions that an exact reproduction of any particular mixture is 
perhaps more difficult than might at first be supposed. Furthermore, 
according to Wolff,* the iron peroxidase is very specific in its action, 
its specificity being determined by the other substances which may be 
present. Certain iron salts or combinations of such may oxidize one 
phenol and be incapable of oxidizing any other. 

The results obtained in experiments on the oxidation of such sub- 
stances as dextrose must be interpreted carefully, as Levene and 
Meyer ^ have pointed out. They showed that in a sugar solution the 
reducing power of which had been lowered by the combined action of 
muscle plasma and pancreatic extract the reducing power was re- 
stored to its original height by boiling under a return condenser for two 
hours in the presence of 1 per cent hydrochloric acid and that a sub- 
stance having the properties of a biosazone could be obtained from 
the above described solution. So that loss of reducing power does 
not necessarily imply destruction of sugar; it may mean a simple 
polymerization. 

The results of the above-mentioned investigations were used in 
planning the experiments that follow: 

THE OXIDATION OF LACTOSE IN BUTTER. 

For the purpose of ascertaining whether lactose which is ordinarily 
present in butter to the extent of about 0.1 to 0.2 per cent is being 
oxidized in cold-storage butter, with the production of substances 
having a disagreeable taste or smeU, any one of several methods sug- 
gest themselves as possible. The lactose present in a lot of butter 
before and after storage may be estimated by any one of the well- 
known methods, or the oxidation products of lactose may be looked 
for in storage butter. 

But the possible oxidation of lactose with the formation of off 
flavors might be brought about with such httle change in the lactose 
content that the ordinary methods of analysis might be inadequate for 
the detection of the change. It was pointed out before (p. 6) that 
very small amounts of some substances are easily detected by the 
senses of taste and smeU and that these amounts are smaller than are 

1 Wolff, J. Relations entre les phenom^nes oxydasiques naturels et artiflciels. Annates de I'lnstitut 
Pasteur, vol. 24, no. 10, pp. 789-797. Paris, Oct. 25, 1910. 

2 Levene, P. A., and Meyer, G. M. On the combined action of muscle plasma and pancreas extract on 
glucose and maltose. Journal of Biological Chemistry, vol. 9, no. 2, pp. 97-107. Baltimore, April, 1911. 



58 CHANGE IN FLAVOR OF STORAGE BUTTER. 

detectable by the best analytic methods of the present time. It was 
also pointed out (p. 15) that the separation of fat quantitatively 
from butter is difficult. This introduces a difficulty in the direct 
estimation of lactose in the butter curd solution. 

For these and still other reasons it was considered advisable to 
avoid attempting to detect directly very minute changes in the 
lactose content of storage butter. The problem was approached 
indirectly. 

On account of the presence of sodium chlorid in butter in amounts 
varying from about 12 to over 30 per cent in the curd solution, care 
must be taken in applying the results of other investigators on 
peroxidases to this problem. It may be that the sodium chlorid is 
without effect. Obviously, only experimental data in which the per- 
oxidase action in the presence of sodium chlorid is studied are directly 
apphcable here. 

One method used in studying (sample No. 25) the utilization of 
oxygen by iron in butter was similar in some respects to that used by 
Horbaczewski ^ and others in their studies of the utilization of atmos- 
pheric oxygen by oxidases of animal tissues. 

Description of samples. — Several gallons of raw milk were obtained 
from a dealer and separated in the cream separator in the laboratory. 
To 8 nters of skim milk sodium chlorid was added, in the proportion 
of 180 grams of sodium chlorid to 1 liter of skim milk. Two and one- 
half hters of this sample (milk No. 25) were transferred to an 8-liter 
flask provided with a rubber stopper through which an inlet and outlet 
glass tube passed to permit the passage of the oxygen gas through the 
sample. Part of the sample was set aside. The milk in the flask was 
the material on which the experiment was made. 

Methods and experimental procedure. — Before beginning the experi- 
ment the lactose content of the sample was determined by its reducing 
power and with the polariscope. In so far as no very great change in 
this quantity was expected, the greatest care was taken to obtain 
accurate results and uniformity of procedure. 

For the gravimetric estimation of lactose the method described on 
pages 42, 48, and 119, BuUetin 107 (revised), of the Bureau of Chem- 
istry was used. It is of course questionable whether the amount of 
copper reduced by a given weight of lactose in milk will be the same 
as that reduced by the same weight of lactose in milk containing 18 
per cent of sodium chlorid. But what was sought was not the abso- 
lute amount of lactose present, but rather the difference, if any, 
between the amount of lactose present before and after a certain 
treatment of the sample. 

1 Horbaczewski, J. Untersuchungen uber die entstehung der Harnsaure im Saugethier organismus. 
Monatshefte fur Chemie, vol. 10, pp. 624-641. Vienna, 1889. 



THEORETICAL CONSIDERATIONS. 59 

Six Gooch crucibles were prepared and used as described by 
Kendall.' 

The weights of the Gooch crucibles after a determination were 
less than their weights before by an amount that varied between 0.4 
and 1.2 miUigrams. The average loss in weight for 19 determinations 
was 0.8 miUigram. Only once did a crucible show a gain in weight, 
0.3 miUigram. 

Although reagents (copper sulfate, sodium potassium tartrate, 
sodium hydroxid) of the highest purity were used, a slight reduction 
was always obtained in blank experiments made on an 18 per cent 
aqueous solution of sodium chlorid. The weight of cuprous oxid 
obtained in the blank determinations (9) varied from 3.6 to 9.1 
milligrams, average 5.9 milligrams. 

The reducing power of sample No. 25 was determined six times in 
duplicate during the course of the experiment. The duplicates dif- 
fered by the following weights of cuprous oxid: 4.3, 0.4, 2.1, 0.2, 0.4, 
0.8 miUigrams. Very nearly 378 milligrams of cuprous oxid were 
always obtained in a determination, from which amount the amount 
of the blank determination was subtracted. 

For the determination of lactose by the polariscope, the method 
given in BuUetin 107 (revised), Bureau of Chemistry, pages 118-119, 
was used : Eight cubic centimeters of acid mercuric nitrate was used 
as the precipitant for 131.6 grams of the sample in a 200 cubic centi- 
meter flask. The filtrate was polarized in a 400-miUimeter tube in a 
Ventzke polariscope. The readings divided by 4 give per cent of 
lactose, if it be assumed that the sodium chlorid was without effect on 
the rotatory power of the lactose. The results are coUected in Table 19. 
Lactose containing one molecule of water of crystaUization calculated 
from the polariscope readings was present to the extent of 4.75 per 
cent. The average weight of cuprous oxid found, 0.371 gram, corre- 
sponds to 0.2577 gram of lactose or 5.19 grams of lactose in 100 cubic 
centimeters sample No. 25. 

The filtrate from the mercuric nitrate precipitation, although per- 
fectly clear at first, soon becomes cloudy and unfit for reading in 
the polariscope. However, readings can be taken witliout interfer- 
ence by cloudiness if they are taken withm one-half hour after filtering. 
The polariscope readings of the filtrates were found to undergo no 
change even after being allowed to remain in the laboratory for two 
weeks. After adding the precipitant and dUutmg to the mark the 
mixture was allowed to stand one hour. It was then filtered and the 
filtrate immediately polarized. Several days after, the filtrate was 
again filtered, a precipitate having formed in the meantime, and 
polarized. In this way several filtrates were repeatedly polarized at 

1 Kendall, Edward Calvin. A quantitative study of the action of pancreatic amylase. Columbia Uni- 
versity, Dissertation, 1910. See p. 10. 



60 CHANGE IN FLAVOR OF STORAGE BUTTER. 

intervals of a few days without in any case detecting an appreciable 
variation in polariscope reading. 

After having determined the lactose in the sample No. 25, its 
specific gravity (in a 25 cubic centimeter pycnometer) was determined. 
Oxygen was then passed through the sample for 72 hours. The oxygen 
was passed from the oxygen tank into an 8-liter flask containing 3 liters 
of 1 8 per cent sodium chlorid solution and then through the milk. This 
was done so that the gas as it slowly bubbled through the milk would 
alter the concentration of the sample as little as possible. While 
the contact between gas and liquid was poor, it is almost certain that 
the liquid was saturated with the gas. About 25 gallons (not quite 
100 liters) of oxygen passed through the sample in the 72 hours. 
Before each determination of lactose the specific gravity of the sample 
was determined and recorded. No significant variations were observed. 

After the 72 hours' passage of the oxygen gas the lactose content 
of the sample was determined and found to have undergone no 
change. This showed, at least in this particular case, that the 
naturally occurring peroxidase in milk could not utilize molecular 
oxygen for the oxidation of lactose. From time to time tests for 
peroxidase were made, using tincture of guaiac and a few drops of a 
dilute solution of hydrogen peroxid. Peroxidase was present in the 
material throughout the experiment. Although always looked for, 
oxidase was not found in the several tests, except once, and that was 
probably due to some unaccountable error. The reagent, tincture 
of guaiac, was not the cause of the unusual positive test. 

To sample No. 25 there was then added 8 grams of ferrous sulfate 
containing 7 molecules of water of crv^stallization (FeS047H20). One 
gram of metaUic iron is present in 4.978 grams of this salt. On adding 
ferrous sulfate to the milk a very strong disagreeable odor was pro- 
duced, suggesting putrefying protein. The odor was undoubtedly 
produced by the action of the iron on the milk, as the sample was 
odorless before the addition. The odor of the material throughout 
the experiment was always carefully noticed. (See p. 64.) The 
quantity of ferrous suKate added was calculated to make one part of 
metallic iron present in 1,000 parts of milk. This is undoubtedly 
much more than is ever present in milk or butter, excluding the case 
where butter is in contact with iron rust, which has gone into solution 
but has not diffused very far into the butter mass. But the amount 
of iron was purposely made very high because the experimental time 
was to be much shorter than the storage period. The milk was allowed 
to stand one day after the addition of the iron, and the lactose deter- 
mined. No change was noticed in this quantity nor was any change 
found after blowing oxygen through the sample for a second period of 
72 hours. The acidity of the sample to phenolphthalem did not 
change during this time, nor were bacteria present during the experi- 



THEORETICAL CONSIDERATIONS. 



61 



ment in numbers sufficient to affect the results. Oxj^gen was passed 
through the sample for 17 days longer with a decided drop m reducing 
power and polarization at the end of that period. This was due to 
bacterial action, as was shown by a suitable examination. 

Table 19.— Action oj iron and oxygen on lactose in milk No. 25. 



No. 



Treatment. 



Reduction. 



Polarization. 



Date of deter- 
mination. 



Weight of 
CuaO. 



Readings 

on Ventzke 

scale. 



Date of 
readings. 



25 
25.1 

25.2? 
25. 2a 
25.2b 



Original sample, containing sodium chlorid; 

age, 7 days 

Repetition on same sample 

After blowing oxygen through for 72 hours and 

letting stand 1 day 

Added ferrous sulfate and let stand 1 day 

Repetition on part of the sample set aside until. 
After blowing oxygen through sample for 

second period of 72 hours, a total of 1-14 

After 10 days slow and 7 days rapid blowing 

of oxygen through sample 



Mar. 28,1911 
Mar. 30,1911 

Apr. 4,1911 

do 

Apr. 7, 1911 



....do 

Apr. 25,1911 



Grams. 
0. 3721 
.3720 

.3703 
.3721 
.3716 

.3692 
.2084 



+19. 
19. 



Mar. 28 
Mar. 30 



Apr. 
Apr. 



Apr. 8 
Apr. 17 
Apr. 25 



During the summer of 1910 three experiments, samples Nos. 1, 2, 
and 3, essentially similar to that on milk No. 25, were made. Au' 
instead of oxygen was used. The results are not appreciably differ- 
ent from those in Table 19. Sample No. 1 was milk, samples 2 and 3 
were skim milk; all three w^ere soured before use in the expermients. 
Only the reduction was determined in these samples which, because 
of the formation of lactic acid, was lower than the reduction in sample 
No. 25. But the reduction did not change appreciably by the action 
of the air blown through the samples. The results were not as uni- 
form as in Table 19, very likely because it is more difficult to sample 
accurately the sour mUk containing particles of casein. After a few 
trials during the summer of 1910 on the best way of obtaining a 
uniform sample for reduction, it was found best to transfer the portion 
of the sample in the pycnometer (25 cubic centimeters) to the volu- 
metric flask for clarification. In this way both the volume and weight 
of the portion used was known. 

THE POSSIBLE OXIDATION OF LACTOSE IN STORAGE BUTTER BY A 

PEROXID. 

. It was stated before (p. 56) that there are at present no reasons for 
supposmg that peroxids are present to any appreciable extent in 
butter. This statement is not free from assumption, for there is a 
possibility of the slow formation of an organic peroxid in butter. 
A review of the literature on the subject and an interesting discussion 
of the formation of organic peroxids by direct combination of the 
compound with molecular oxygen has been made by Kastle.^ The 



1 Loc. cit. 



62 CHANGE IN FLAVOR OF STOEAGE BUTTEK. 

detection of an organic peroxid in butter at any particular time might 
be practically impossible because of its almost immediate decomposi- 
tion either by the catalase naturally present in unpasteurized cream 
butter or by iron almost always present, even in butter most carefully 
churned, to the extent of three or four parts per million of butter 
(see p. 54 for amounts of iron in butter) and yet the oxidative process 
may be taking place. Perhaps a careful analysis of the gas in butter 
will show whether any of the oxygen is being removed in this way. 
The work on the analysis of the gases in butter has been begun and 
some of the analyses are given on page 37. 

For the reasons just mentioned the fact that no oxidation of lactose 
was detected in skim milk containing iron and through which oxygen 
was passed does not exclude the possibility of the slow formation of 
organic peroxids in butter and their subsequent oxidative action. 

On the assumption that organic peroxids might be slowly formed 
in butter, and that such peroxids might be used by the peroxidase 
present for oxidation, a few experiments were made in which the 
polarization of skim milk was observed before and after the addition 
of hydrogen peroxid, with and without iron. The data are sum- 
marized in Table 20. One liter of each mixture was prepared from 
which portions were transferred to a 200 cubic centimeter flask for 
clarification and polarization. 

It is evident that in these mixtures containing hydrogen peroxid 
and iron (Nos. 18, 19, and 23) there was a very appreciable lowering 
in the polarization. This lowering probably was not due to the easy 
reducibility of the mercury by the lactose, because in those filtrates 
containing hydrogen peroxid but no iron (Nos. 17 and 21) there was 
no lowering, although these filtrates contained unprecipitated mer- 
cury as well as the others. While there may be other possibilities, 
the most reasonable tentative conclusion to be drawn from the results 
of Table 20 is that the lactose was oxidized by the action of the 
peroxid and iron, and if there were peroxid formation in butter such 
oxidation of lactose might take place there. The experiments detailed 
in Table 20 were not expected to be conclusive; others are undoubtedly 
necessary. 



THEORETICAL CONSIDERATIONS. 



63 









*t 


OOtO lO 








■^ 


C^l O CO cr. 


, 








oioico 










cid dci 


























i 










a 










% 
































































(M 


Cl .-< CI w 








CO 






















» -.C CO 








^ 
<& 


d d d cc 










oioico 


























p:, 












~ 
































p^ 










O 




















J2 


ci o eo (=■ 
d d d -p 








o 












end CO ,5 








pq 










X! 


^H c/; 


















t^ 






















^ o CO c; 


















d d d I-- 














.d 






^I, 1 


00.-HO 














oid lo 
















-r i~ 














OIC CO CO 










X d d oi 




P=H(i, . 






















— IM '. 












fO 


M CO I^ C 












d d -.dir 








CO 


Ort Tt> I- 
















d d d <m' 












X2 

P-H 








SS§5 


CO CO CO I^ 

ddco t 




«OJ ' 


OIM IOC 






c c d 


.-< ^H 




■g-s ■§ 


d d d -o 




a oj 03 


+ 


























f^p^ 




ic 






























CO 




-- - 


C 
























-* X"-* CO 


c3 


•^ 




















d d d -o 


T3 

s 


% 






,, 


OC 


o- 












c3 


"O T^ 


1 «— t f—i .-H .— 












Precipit 

tered. 

Let star 

Polarize 


ii 6 d d c 




C-1 -f -tf 


c-i ■* >* ■* 


03 




xJxi x; 


+ 










s 


: a 


























TOS. 

one. 
one. 
one. 

180 




•a 


I'd 

; ft 

3g 
















T35 


ezziz; 




o 


W c3 




. d -<' oi CO 




■g-g 






03 


C3 P 




i d d d d 










-■^■rt a R 












recip 
tere 

et St; 

efllt* 
ized 


|z;2;2;!z 












OO OC: 




03 




3 


^^rt rt .- 




Ph hJ« 


03 




o 


O 






H c? ^ a 






H 








2 ai d^^ 
5 C C - 












Fei 

acel 

po 

der 


goo 


JJ 








hii 


^ o o 














1 


10 per 

ferr 

chlo 

solut 


o^;Z 






ad 

CO o 


Grams. 
180 
180 
180 
180 




OOOU- 












c:; 05 -o x 










o 


B 


0-* 


£ 




c 


gdgo; 


s 


el 








S, 


.i!5 Cb; C 


.2 

o 
ft 

s 

o 


^ 


Cj 


_ 6 




03 


o o O 
^ Z Z 


Hydrogen 
peroxid 3 
per cent 
solution. 


C.C. 

None. 
400 
400 
300 


o 

o 

ft 

a 


Hydrogen 
peroxid 3 
per cent 
solution. 


r. c. 
None. 
500 
None. 


'aJ-d 




o 

O 


^ 3 

a^d 


Jill 




C 






fiSg 






M a 








■3 P.--^ 






OQ 3 














-45 t~ 'JO O- 




o 


^ ;^ J1 CO 










P 






rSi 








M 





64 CHANGE IN FLAVOR OF STORAGE BUTTER. 

Lob and Pulvermacher's observation (p. 56) that dextrose is much 
more easily oxidized than sucrose, and that the sucrose apparently 
can oxidize only as fast as it is first inverted, suggests the desirability 
of further experiment along the lines of the work on sample No. 25, 
but in which the sample is soured before being used in the experiment. 
In the presence of lactic acid (or its combination with casein) it is 
very probable that the lactose present would be inverted, even if very 
slowly. Then the iron and oxygen would have the entire storage 
period of several months to bring about the slight chemical changes 
presumably sufficient to give butter an "off flavor." 

ODORS PRODUCED IN MILK BY THE ADDITION OF IRON SALTS. 

In our experiments the production of substances having a dis-* 
agreeable odor and taste was the most important part of the work. 
No chemical change, however pronounced, that presumably did not 
affect the flavor of butter was of more than incidental interest. 
For this reason the odor of the experimental material was always 
carefuUy noted. 

It was state'd before (p. 60) that on addmg ferrous sulfate to milk 
containing salt, a very strong, nauseating odor was produced. This 
observation had been made before in mixtures of milk, hydrogen 
peroxid and ferric chlorid, but the odor was not produced every time 
that ferric chlorid and hydrogen peroxid were added to milk. It 
was obviously desirable to know whether iron salts could produce 
undesirable odors in milk and whether the experimental conditions 
under which such odors were produced were in any way similar to the 
conditions in cold storage butter. 

After several trials it was found that ferrous salts added to milk 
in the proportion of 1 part of metallic iron to 1,000 parts of milk 
would result in the production of a very powerful odor. The method 
of the experiment was very simple as the following example shows : 

Several liters of raw milk as obtained fresh from the dealer were 
separated. Sodium chlorid was added to the skim milk, anywhere 
from 18 per cent to saturation, or the sodium chlorid may be omitted 
altogether. The sodium chlorid serves both as a preservative and as a 
normal constituent of butter curd solution. Skim milk was used 
because by eliminatmg the fat and fat soluble substances the sub- 
stances causing the odor could be better determined. The sample 
of salted skim milk could be used at once or after several days' stand- 
ing at room temperature. Portions of 300 cubic centimeters each 
were transferred to 1-liter Erlenmeyer flasks. To each flask there 
was then added a calculated quantity of the metal salt. The follow- 
ing salts were used in the several experiments; some of them were 
used very many times: Ferrous sulfate, ferrous ammonium sulfate, 



THEORETICAL CONSIDERATIONS. 65 

ferrous lactate, ferric acetate, ferric chlorid, and to a lesser extent 
some salts of the following metals: Copper, manganese, aluminium, 
and lead. 

In no case was any odor produced by any metal salt other than 
iron. Ferrous salts almost always, ferric salts very seldom, pro- 
duced an odor in the milk. The quantities used were calculated to 
make 1 part of metal present in 1,000 parts of milk. When this 
quantity of a ferrous salt is used the odor becomes very powerful 
in a few minutes. However, 1 part of ferrous iron in 50,000 parts of 
milk could easily be shown to produce an odor plainly perceptible by 
several persons to whom no information had been previously given 
regarding the nature of the samples to be smelled. The odor develops 
slowly when the amount of iron is small. For 1 part of iron to 50,000 
of milk an hour should be allowed. 

It seems that the odor is developed at that time when the color of 
the mixture changes to the more highly colored ferric salt. 

Although the odor strongly suggests putrefying protein or hydrogen 
sulfid, tests made for hydrogen sulfid were negative. The tests were 
made by passing a current of air through milk in which an odor had 
been developed by the addition of ferrous iron, and then through an 
alkaline solution of lead acetate. 

It is doubtful whether the odor came from the fat, because skim milk 
was used, and it was doubtful whether lactose was the cause. It 
seemed probable that protein was being acted upon by the iron. In 
one experiment in which solutions of egg-white and of egg-yolk were 
used the same results were obtained as before, i. e., the addition of 
iron salts (ferrous sulfate and ferric chlorid) resulted in the pro- 
duction of a strong odor. Whether protem was acted upon in the 
experiments or not, is difficult to say. 

On looking through the literature on the oxidation of proteins, 
very little was found that would throw light on the oxidation of 
protein by means of comparatively mild oxidizing agents. In most 
of the researches the protein was broken down completely by the 
reagents used, and the work was done for the purpose of studymg either 
the products of the oxidation or the various oxidation stages through 
which the protein goes durmg the course of metabolism. No records 
were found in which the protem oxidation was studied for the par- 
ticular purpose of observing whether odoriferous substances were 
formed. The oxidation of protein in storage butter (if it occurs at 
all) is probably very mild. There seems to be no apparent change in 
the quantity of protein in butter before and after storage. 

However, two researches were found in the literature which were 
highly instructive. 



66 CHANGE IN FLAVOR OF STORAGE BUTTER. 

Neuberg and Blumenthal ^ studied the oxidation products of 
gelatin, using ferrous sulfate and hydrogen peroxid. In the distillates 
from 2 kilogi'ams of gelatin they isolated and identified isovaleralde- 
hyde. Other volatile products were also formed. Orgler ^ repeated 
some of the work of Neuberg and Blumenthal, using crystalHzed 
Qgg albumin, copper sulfate, and hydrogen peroxid. Acetone was 
detected and identified in the distillates from such a mixture, which 
distillate, according to Orgler, had a strong fruity odor. 

It follows that if the ferrous salts used in the production of an odor 
in milk act on some milk protein with the formation of aldehyde or 
ketone substances, it ought to be possible to take milk (containing salt) 
add a ferrous salt, and obtain from it by distillation some of the sub- 
stances mentioned by Neuberg and Blumenthal and by Orgler. Two 
experiments were made which, while not so conclusive as to make 
further experiment umiecessary, gave results so much in accord with 
expectations that there is little doubt but what the ferrous sulfate 
used in the experiments caused the formation of substances which 
gave the iodoform test. Beyond this theu- chemical nature was not 
investigated. 

THE PRODUCTION OF lODOFORM-REACTING SUBSTANCES IN MILK BY 

FERROUS IRON. 

To an ordinary 2-liter side-arm distillation flask 1 liter of fresh 
skim milk was transferred (sample 42.2). The sample contamed 200 
grams of sodium chlorid to 1 Hter of skim milk. It was quickly 
brought to a boil and 10 cubic centimeters distilled over. This was 
tested with sodium hydroxid solution (1:3) and iodin solution 
without heating for substances giving the iodoform test.^ 

The test was positive. The above procedure was repeated on 
some of the same sample of milk under the same conditions, except 
that 5 grams of the crystalhzed ferrous sulfate (Fe 1 : 1 000) was 
added just before distilling. The distillate gave a much stronger 
iodoform test; that is, there was a very appreciable immediate pre- 
cipitate of iodoform. A second sample of skim milk was distilled 
as before. This sample (No. 37x) was obtained from the same 
dealer as the previous sample (sample 42), and after being skimmed 
they probably did not differ very materially in their composition. 
At the time of the" experiment this sample (37x) was raw skim milk 
containing 30 per cent of sodium chlorid (300 grams of salt to 1 liter 
of milk) and 1 part of iron as ferrous sulfate to 5 000 parts ol mUk. 

1 Neuberg, C, and Blumenthal, F. Uber die Bildung von Isovaleraldehyd und Aeeton aus Gelatine. 
Beitrage zur Chemischen Physiologie und Pathologie, vol. 2, no. 5-6, pp. 238-250. Braunschweig, May, 
1902. 

2 Orgler, Arnold, tjber die Entstehung von Aeeton aus krystallisiertem Oralbumin. Beitrage zur 
Chemischen Physiologie und Pathologie, vol. 1, no. 10-12, pp. 583. Braunschweig, January, 1902. 

3 MulUken, Samuel Parsons. Identification of Pure Organic Compounds. See vol. 1, p. 166. 



THEORETICAL CONSIDEEATIONS. 67 

The salt and iron were added on the same day the sample was received. 
After remaining at room temperature in the laboratory I'or 12 days, 
during which time several htei-s were used for other purposes, 3 
liters of this sample were transferred to a 7-liter bottle and oxygen 
was blown through it slowly for 4 days (96 hours). One hter of 
this was distilled. The distillate (10 c. c.) yielded more iodoform 
than either of the other two. Judging by inspection, on distilling 
fresh skim milk a veiy small but distinctly perceptible quantity of 
lodoform-reacting substance was obtained; fresh skim milk and 
iron yielded more, and skim milk and iron first saturated with oxygen 
yielded most. There could be Uttle doubt about the relative amounts 
of iodoform, but the experiment was repeated on portions of the 
same samples as before for the purpose of estimating quantitatively 
the iodoform obtained. 

Each distillation was continued until six 10 cubic centimeter 
portions of distillate were obtained. Iodoform tests on these were 
made by adding to each portion contained in a test tube 10 drops 
of sodium hydroxid solution (1:3) and sufficient iodin solution to 
insure a slight excess. In every case precipitates of iodoform were 
obtained almost immediately and without the aid of heat. Very 
Httle iodoform, if any, was obtained in the last 10 cubic centimeters 
of distillate. 

The iodoform was washed first by decantation, then transferred 
to filter papers and washed till the filtrates gave only a very faint 
cloud with silver nitrate solution. Some of the iodoform was, of 
course, lost through solution in the wash water, but the relative 
amounts in this case rather than the absolute were just as desirable. 
The amount lost in this way probably was small compared with the 
amounts present. The blank determination (No. 42.3) was lost. 
It contained a distinctly perceptible precipitate, but too small in 
amount to compare with either of the other two. For the estima- 
tion of the iodoform a method given in the Pharmaceutical Journal, 
page 555, volume 82, 1909, was used. The iodoform on the filter 
papers was dissolved in alcohol and ether and the solutions received 
in 300 cubic centimeter Erlenmeyer flasks. To these flasks and to 
controls 1 cubic centimeter nitrous acid (fuming nitric acid) and 
50 cubic centimeters approximately tenth normal silver nitrate 
solution were added. The mixtures were heated on the steam bath 
over night. The silver stiU remaining in solution was estimated with 
standard ammonium sulfocyanid solution, using ferric ammonium 
sulfate as indicator. 

The distillate from the fresh milk containing feiTous sulfate (No, 
42.4, Fe 1 : 1,000) yielded 15.4 milligrams of iodoform; the distillate 
from the 12 days' old milk containing ferrous sulfate (No. 37x, Fe 1 : 
5 000) and saturated with oxygen gave 54.7 milligrams of iodoform. 



68 CHANGE IN FLAVOR OF STORAGE BUTTER. 

These amounts of iodoform correspond in the titrations to 1.37 
cubic centimeters in the first and to 4.88 cubic centimeters of silver 
nitrate solution (N/11.7) in the second determination. 

While these results do not prove that the iodoform was obtained 
from oxidation products of milk protein, they do prove the possibility 
of such oxidation. By distiUing such mixtures under low pressure 
and at low temperature to remove the possible objection that the 
temperature of distillation is not the temperature at which chemical 
changes take place in storage butter, the identity of the iodoform- 
reacting substances could without doubt be ascertained. 

Wliether the small amounts of iron ordinarily present in butter 
(see p. 54) can slowly bring about the same kind of a change that 
larger amounts of iron bring about in milk in a very much shorter 
time is to be determined by future investigation. 

SUMMARY. 

The failure of previous investigators to find evidences of pro- 
teolysis in cold-storage butter may have been due to difficulty in 
obtaining proper precipitations in the curd solution. 

Methods of analysis have been perfected which permit the use of 
large samples and show the first stages in the proteolysis. This 
method gave no evidence of an increase in soluble nitrogen in butter 
on long standing at 0° F., even when the conditions of the manu- 
facture were most favorable to such changes. 

Buttermilk from sweet unpasteurized cream and from sweet 
pasteurized cream when preserved with 18 per cent sodium chlorid 
to correspond to butter-curd solution showed no proteolysis during 
a long period in cold storage. 

Bacterial enzym held in cold storage in milk containing 18 per 
cent of sodium chlorid gave some evidence of proteolysis. The action 
of pepsin and trypsin under similar conditions was not completely 
inhibited. 

Butter made from sweet pasteurized cream keeps much better 
than butter made from similar cream without pasteurization, but 
the changes in the unpasteurized cream butter can not be repro- 
duced by reinoculating the pasteurized cream with the bacteria of 
the cream before pasteurization. 

By means of specially designed apparatus exact analysis was 
made of the gases contained in butter. About 10 per cent, by 
volume, of fresh butter is gas consisting approximately of nitrogen 
(by difference) 33 per cent, oxygen 20 per cent, and the remainder 
of gases absorbable by sodium hydroxid. The oxygen was materially 
less after storage. 

The addition of iron to the cream even in as small an amount as 
one or two parts per million parts of cream has an influence on the 



SUMMARY. 69 

flavor of tlie butter. This work gives nothing to show that the 
nature of the flavor is appreciably changed, but the rate of develop- 
ment is accelerated. 

The cream may take up iron in quantities sufficient to affect the 
flavor from rusty cans or even from the exposed boltheads or other 
metal parts of the churn. 

The action of copper is similar but perhaps more intense. 

It was found that in milk to which 18 per cent sodium chlorid 
had been added there was no change in the lactose when iron was 
added and a current of oxygen passed through the milk for 72 hours. 

A strong odor may be produced in milk by the addition of small 
amounts of iron salts. The ferrous salts are more active than the 
ferric salts. 

The iodoform test is much stronger in distillates from milk con- 
taining ferrous sulfate. 



ADDITIONAL COPIES of this publication 
-^ may be procured from the Superintend- 
ent OF Documents, Government Printing 
OflBce, Washington, D. C, at 10 cents per copy 




LIBRARY OF CONGRESS 



000 896 162 1 



